FI110433B - Alpha-amidating enzyme - obtd. from rat medullary thyroid carcinoma and used for prodn. of peptidyl amide cpds. having an amino gp. in place of a C-terminal glycine - Google Patents

Abstract

An enzymatic compsn. is claimed comprising at least one alpha-amidating enzyme (AAE) capable of catalysing, in the presence of oxygen and a reducing agent, the conversion of a peptidyl cpd. having a glycine residue at least at the C-terminus of its peptide chain to a corresponding peptidyl amide cpd. having an amino gp. in place of the C-terminal glycine, the enzymatic compsn. being sufficiently pure in AAEs to exhibit a specific activity of at least 25 mll per mg of protein present and the enzymatic compsn. being sufficiently free of proteolytic activity capable of degrading at least one of the peptidyl cpd., the corresponding peptidyl amine and the AAEs to allow the compsn. to catalyse the conversion when the peptidyl cpd. comprises L-amino acids. Also claimed are immunoaffinity columnss for purifying the AAE and method for prodn. of AAE compsns. using DNA sequences coding for AAE.

Description

110433

DNA sequence encoding an alpha amidation enzyme. host cell and process for preparing an amidated peptide This application is a continuation of U.S. Patent Application No. 655,366, filed September 27, 1984, which is incorporated herein by reference in its entirety.

This invention relates to alpha-amidating enzymes, to the preparation of alpha-amidating enzymes, and to their use in the production of alpha-amidated enzymes by the action of enzymes on glycine expanded substrates. In certain preferred embodiments, the alpha-amidation enzyme encoded by the DNA sequence of the invention may be used in the preparation of useful alpha-amidated hormones and agricultural and pharmaceutical products containing calcitonin, growth hormone releasing agents, calcitonin gene related peptides and other alpha products.

The intracellular processing (cleavage and / or functional group transformation) of precursor forms of natural proteins and their translation from nucleic acid coding sequences have been described in detail.

In general, mammalian cells and other eukaryotes may undergo certain post-translational treatments, whereas prokaryotes may not. Certain procyotes,.:: Such as R coli are widely used as hosts for the preparation of mammalian proteins. '! * by the technology of ruminant DNA (rDNA) because they can be easily grown

• · I

• · · * fermentation methods and because they are genetically well characterized. However, mammalian proteins produced by genetic recombinant technology require some type of post-translational processing, and this must often be accomplished using complex chemical in vitro procedures. which are expensive in large-scale manufacturing.

• · »· · · · ·

One way to treat activity is to use specific amidation of the carboxyl-terminal amino acid "·" of the protein. Many naturally occurring hormones and peptides include; * 30 such a variation, which is often necessary if the protein is to be biological • · * *. * active. One example is kaisitonin, where the substitution of a natural amidated proline:. 'With a non-amidated proline residue produces biological activity of 3,000-.

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fold decrease.

2 110433

The agent that affects this C-terminal (alpha) amidation recognizes a glycine residue immediately following the amino acid to be amidated (R-X-Gly, where R is the protein backbone, X is the amidated residue, and "Gly" is the glycine residue). Glycine loh-5 lanes and actually transmits the amino moiety to the last amino acid and amidates it.

The first researchers to provide an approximate Mole of alpha amidation enzyme were Bradbury A.F. et al., Nature, Vol. 298, 1982, pp. 686-88. Using 10 Sephadex G-100s, they suggested an apparent minimum molecular weight of about 60,000 Daltons.

In the following studies, such an enzyme has been suggested to have a molecular weight of 60,000 to 70,000 daltons (as measured by gel filtration chromatography). See. Husain, I. and Tate, S.S., 15 FEBS Letters, Vol. 152 # 2,1983, pp. 277 - 281; Eipper, et al., PNAS Vol. 80, 1983, pp. 5144-5118; Gomez et al., FEBS Letters, Vol. 167, # 1, 1984, pp. 160-164 and Kizer, J.

S., et al., PNAS, Vol. 81,1984, pp. 3228 - 3232.

Eipper et al., PNAS, Vol. 80,1983, pp. 5144-48, have suggested that in addition to molecular oxygen, two cofactors are required for maximal enzyme amidation activity | : there. These are ascorbic acid and copper (II) ion.

The chemical reaction that results in the amidation of the carboxyl terminus of the peptide requires a source of the amino group. Bradbury, A. F., et al., Nature, Vol. 298,1982, pp. 686 - 688, • »* i. * 25 showed that glycine is cleaved and releases the last of the amino acid to the previous amino acid to amidate it. Other researchers have confirmed the need for glycine as an amino acid donor.

• · · • · * • · T Landymore, A. E. N., et al ·? BBRC Oh. 117, # 1,1983, pp. 289-293, showed that D- • ♦ '·' · '30 alanine could also act as an amino donor in the amidation reaction. Subsequently, Kizer * · · et al., PNAS, Vol. 81, 1984, pp. 3228-3232, showed two distinct enzyme activities in rat brain capable of catalyzing an alpha-amidation reaction. The high molecular weight type (70,000 daltons) has a specificity limited to glycine at the carboxyl terminus of the substrate. The lower molecular weight enzyme accepts a substrate containing β-alanine at the carboxyl-terminal amino acid.

5 Bradbury, A.F. and Smythe, D.G., BBRC, Vol. 112, # 2,1983, pp. 372-377, disclosed an alpha-amidating enzyme extracted from porcine pituitary and specifically purified to a pH of about 7.0. Eipper, B.A., et al., PNAS, Vol. 80, 1983, pp. 5144-5188, confirmed these results and suggested that the alpha-amidating enzyme partially purified from rat pituitary had a pH optimum of 7. They also found that the enzyme activity was ale-10 rapidly at pH values below 6, 5 or more than 7.5.

The extracts and partially purified enzyme mixtures disclosed in all of the above publications (incorporated herein by reference) contain additional proteolytic enzymes which may cleave any substrates or products, as well as the Alfani amidation enzymes themselves, thereby slowing down the amidation of peptides and polypeptides by such enzymes. produced by recombinant DNA technology.

In general, all previously measured amidation activities of others were based on the conversion of D-20 substrates such as tripeptide, DTyr-Val-Gly-COOH to D-Tyr-Val-i: CONH2. Of the two possible configurations ("D" or "L"), naturally occurring # '': biologically important amino acids occur in the "L" form. However, the "D" form used by these other investigators was required to counteract the presence of irrelevant proteolytic enzymes in the impure amidation enzyme preparations used by these investigators. These irrelevant enzymes may have a co-elaborated proteolytic effect on the L-amino acid substrate while having little effect on the D-substrate. Researchers burdened with proteolytic and other impurities in their enzyme used an unnatural "D" substrate for impurities *; * to avoid some of the effects of guarantees. To date, it has not been possible to demonstrate: * 30 countries that their enzyme preparations can efficiently amidate any physiologically relevant substrates, i. L-substrates for their conversion into biologically active alpha-amidated L products.

• * • *> · • · <• · 4 110433

As shown herein, the formulations of the present invention are capable of efficiently amidating L-substrates and exhibiting activity of D-substrates that is 60 to even 1000 times greater than the highest activity known to those skilled in the art.

5

Several sources have provided enzyme preparations capable of amidating the carboxyl terminus of peptides and proteins. For example, Bradbury. A.F., et al., Nature, Vol. 298, 1982, pp. 686-688 (herein incorporated by reference in their entirety) disclose the activity of the alpha amidating enzyme in the porcine pituitary. The enzyme-containing preparation of porcine pituitary gland 10 is capable of converting peptides terminating in glycine to the corresponding desglycine peptide amide. Bradbury et al. acknowledge, however, that the preparations do not amidate peptides or polypeptides which have been purified from natural sources: "Dtyrosylvalyl-15 Glycine to the corresponding dipeptide Amide Dtyrosylvaline Amide ... The D-tyrosylvaline conferences stability against degradation by aminopeptidases present in tissue homogenates ... Control experiments showed that when synthetic ... D-tyrosylvaline Amide was slowly degraded. the formation of the dipeptide Amide by the pituitary enzyme is followed by its Destruction by other enzymes 20 present in the pituitary extract. " (p. 686). (An analytical system for the detection and evaluation of amidating activity in the pituitary was obtained by studying the ability of enzyme preparations to convert the synthetic tripeptide D-tyrosylvalylglycine to the corresponding dipeptide · · · • 1-diamide, Controlled I · ·: 25 studies showed that when synthetic ... D-tyrosylvalinamide was incubated under similar conditions, it was degraded slowly, thus inhibiting the formation of dipeptide amide by the pituitary enzyme. digestion with other enzymes present in the pituitary extract).

· '·' Thus Bradbury et al. acknowledge that the formulations disclosed contain other proteolytic products

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'' Enzymes that degrade peptides or polypeptides and that the non-naturally occurring D-1 .: tyrosine residue had to be used to minimize such degradation.

»1 1 • · 5 110433

Further, Bradbury et al. teach the use of homogenized or subcellular fraction followed by gel filtration chromatography to purify the amidation enzyme, which is undoubtedly still impure due to proteolytic enzymes.

5

Husain, I. and He you. S.S., FEBS Letters, Vol. 152, # 2,1983, pp. 277-281 discloses alpha-amidating activity present in bovine pituitary neurosecretory granules.

io Eipper et al., PNAS Vol. 80,1983, pp. 5144-51148 disclose alpha-amidating enzyme activity present in the anterior, middle and posterior segments of the rat pituitary and in the middle section of the bovine pituitary. However, this reference only teaches the use of a synthetic D-Tyr-Val-Gly substrate to identify the impurity of the manufactured products for alpha-amidation activity.

15

Gomez et al., FEBS Letters, Vol. 167, # 1,1984, pp. 160-164, have determined that the rat hypothalamus also contains alpha-amidating activity.

Bradbury, AF, Smythe, DG, Peptides Structure and Function: Proceedings of the Eighth American Peptide Symposium, pp. 249-52 (1983), published by Hruby, VJ and Rich, DH, disclose the presence of alpha-amidating enzyme activity in the rat shield. - • ·. : in the gland.

I · ·> * • · * = '' Mains R.E. et al., Endocrinology, Vol. 114,1984, pp. 1522-1530, have reported that the * · ·: · '25 anterior pituitary cell strain (ATT-20) contains alpha • · * * *' 'amidating enzyme activity, which is clearly reduced in culture.

Glands or organs known to contain amidated peptides may contain ent! 'An enzyme capable of catalyzing an amidation reaction. For example, O'Donohue T. L., et al., Peptides 3, 1982, pp. 353-395, have suggested that lower life forms such as • · * ·; Dog shark (Scalus acanthi as) containing amidated peptides in pituitary extracts. Schel-1 '. Ler, R. H. et al., Cell, Vol. 32,1983, pp. 7-22, have provided amidation signal peptides

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'· * · Presence in the sea snail, Anlvsia. Despite the apparent presence of this activity everywhere in nature, little information has been published on the physicochemical properties of the enzyme. This may be due to very low levels of the enzyme in these neuroendocrine organs.

The presence of amidated peptides in a particular tissue may not be co-branded with high levels of the alpha-amidating enzyme. For example, rat rat adrenocortical tissue contains high alpha-amidating activity but no known substrates (Eipper et al., PNAS 80, 5144-5188, 1983). Rat rat pituitary tissue contains amidated peptides (oxytocin and vasopressin) but has very low alpha-amidating activity (Eipper et al., Endo 116,2497-2504,1985). Therefore, until the tissues are tested for alpha-amidating activity, the presence or potential levels of the enzyme cannot be predicted.

It is an object of this invention to provide alpha-amidating enzyme compositions which can efficiently produce alpha-amidated products even from substrates containing L-amino acids, such as peptide or polypeptide substrates purified from natural sources or prepared by recombinant DNA technology.

It is still a task to provide efficient and inexpensive methods for preparing the enzymatic compositions.

• · • · · ·. ' *: These further produce monoclonal antibodies specific for the alpha-M · V amidation enzyme, and immobilized antibodies, purification resins,:. Immunoaffinity columns and the like employing these antibodies to efficiently purify the alpha-amidation enzyme.

• · · • · · · ·

It is still a task to provide genetically engineered host cells that are stable. * enter high alpha amidation enzyme expression.

It is a further object of the present invention to prepare alpha-amidated products from substrates; · * Containing a C-terminal glycine residue by reacting the substrate in the presence of an enzyme composition.

• I · 7 110433 I These and other tasks are explained in the following detailed description. According to the invention, the applicants provide novel alpha-amidating enzyme compositions of sufficient purity having a specific alpha-amidating activity of at least about 25 mU / mg protein, and in particular greater than 50 or more than 150 mU / mg protein. All units of specific activity presented herein are based on the conversion of dansyl-D-Try-Val-GlyCOOH to dansyl-D-Tyr-Val-CONH2. One mU is defined as the amount of activity required to convert one nanomolar (nmole) of dansyl-D-TyrVal-Gly-COOH to one nmole / min of dansyl-D-Tyr-Val-CONH2.

At 37 ° C and pH 7.0 in the presence of ascorbate ions at a concentration of 3 mM (based on the entire reaction mixture including enzyme, substrate, cofactors, etc.), in the presence of a molar excess of molar acid relative to the substrate and of copper (II) ions. , which can maximize activity (usually about 2 μΜ, depending on the purity of the enzyme).

The alpha-amidating enzyme encoded by the DNA sequence of the invention, the peptidylglycine-alpha-amidating monooxygenase, is capable of catalyzing the conversion of a peptidyl compound having at least a C-terminus of the peptide chain to the corresponding peptidylamide terminal compound of glycine. As used herein, the term "peptide chain" refers to a polypeptide having at least two amino acids linked together by a peptide bond. The term "peptidyl compound" means. : a compound having a peptide chain. The term "corresponding peptidylamide" refers to the reaction. ': A thio-product which replaces an amino group in the peptide chain in place of the C-terminal glycine.

Preferably, the alpha-amidation reaction is carried out in the presence of oxygen and a reducing agent. Suitable «* ·« · * :; Reducing agents include, for example, ascorbic acid, ascorbic acid salts, dihydroxy fumarate, metal cyanide compounds and tetrahydropterin. Certain cofactors have been found. . promotes the reaction and / or slows down the degradation or inactivation of the enzyme.

! .. 'These cofactors include: catalase, ethanol, potassium iodide and copper (II) ions. The purified enzymatic compositions obtained by the process of the invention are, in particular, sufficiently free from proteolytic activity capable of degrading the products or starting materials of either the alpha-amidating enzyme or the alpha-amidation reaction. The 'cystic compositions can catalyze the alpha-amidation reaction even though the substrate and product are composed of L-amino acids. The proteolytic activity of the amidation reaction mixture cannot directly reflect the protease concentration. Activity, even at high protease concentrations, can be inhibited, for example, by the action of various inhibitors. The lack of proteolytic activity, which increases the commercial and practical desirability of the enzymatic composition for use with L-amino acid substrates, does not in any way diminish the utility of the composition with non-L-amino acid substrates.

As detailed in the detailed description, the Applicants have purified a number of specific proteins having substantial alpha-amidating activity. As used herein, the term "alpha-amidating activity" refers to an activity which tends to leave only the amino group previously occupied by the C-terminal glycine of the substrate peptidyl compound. Such substitution may require cleavage of all but the amino group of glycine so that only the amino group remains in the position previously occupied by the entire glycine moiety. As used herein, "alpha-amidating enzyme" refers to a composition or compound having alpha-amidating activity and their active homologues and fragments.

The enzymatic compositions obtained according to the present invention can be purified to homogeneity. As used herein, "homogeneous" refers to enzyme preparations having a single, well-defined band following electrophoresis with sodium | · With dodecyl sulfate / polyacrylamide gel and having one single amino acid mixture. ': Quantity data according to common sequencing methods.

• »i .: The α-amidation enzyme encoded by the DNA sequence of the invention, which has been purified to homogeneity, has a specific enzyme activity of more than about 1500 mU / mg * · * protein.

The enzymatic compositions containing the enzyme encoded by the DNA sequence of the present invention can be used to prepare useful alpha-amidated products using the enzymatic compositions and. * '·; · 'To catalyze alpha-amidation of such products. There is provided a substrate which • '. Is a peptidyl compound having a glycine residue at least at the C terminus of the peptide chain, where:. '·; Substitution of the amino group on the C-terminal glycine residue affords the desired product.

The substrate is reacted, in particular in the presence of oxygen and a reducing agent, in the presence of an enzymatic composition of the present invention, in particular in the presence of oxygen and a reducing agent, for a sufficient time to convert the peptidyl compound to the corresponding peptidyl amide. The conversion commences substantially upon initial contact with the substrate and the enzyme, but the reaction rate varies substantially according to pH, temperature, substrate identity, cofactor concentration, and other parameters that can be adjusted in a known manner to optimize the reaction. The reaction is usually allowed to proceed for a period of time selected according to the desired conversion rate (substrate to granny product). In preferred embodiments, cofactors such as those mentioned above are used to promote the reaction and / or to enhance or enhance the activity of the enzyme.

A variety of useful products, including natural hormones and the like for which alpha amidation is advantageous or necessary, can be prepared by reacting glycine-extended peptidyl compounds in the presence of alpha-15 amidation enzyme compositions obtained according to the present invention. These products, which can be used, for example, in agriculture or in the treatment of diseases characterized by hormonal deficiencies, include: various calcitonin, growth hormone releasing factors, calcitonin gene related peptides and the like. The hormones referred to herein, such as the aforementioned calcitonin, growth hormone releasing factors and calcitonin gene related peptides, contain C-terminal amide proteins expressing the characteristic activity of said hormone, as will be apparent to one skilled in the art.

For example, calcitonin contains all species that express an upregulation of calcium uptake into bone, which is characteristic of known calcitonin. The various proteins presented herein *

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! *! * Homine species homologues are included within species definitions and the nucleotide sequences herein are homologous sequences in which substitutions, additions> 19 '' 'or deletions do not substantially affect the function of the sequence presented. Preferably, at least 40%, and in particular 50%, of the amino acids of the peptide correspond to those disclosed. Of course, with respect to the nucleotide sequences of the nucleotide »I» »» »i .. ', the codons may be substituted by equivalent codons> · * I' which encode the same amino acids.

I · I * »I I * * * 30 I 1 * t * '·; * 'An enzyme composition containing the enzyme encoded by the DNA sequence of the invention. · Ability of nuclei to catalyze an alpha-amidation reaction even with L-amino acids * »I> I t '. * 'With substrates enables the efficient and cost-effective preparation of these products using substrates that have been purified from natural sources or made by recombinant DNA technology.

In certain preferred embodiments, the alpha-amidation reaction can be facilitated by immobilizing the enzyme on a solid carrier which is insoluble in aqueous media and resisting degradation under the reaction conditions, and by directing the substrate through the immobilized enzyme, particularly in the presence of suitable cofactors. Here, "immobilization" refers to the binding of an enzyme to a substrate. Suitable carriers for this purpose are, for example, a regulating pore glass or an activated absorbent such as sepharose activated with cyanogen bromide. Enzymes immobilized in this manner can be reused by removing the reaction mixture from a solid support that retains the enzyme for future use.

Applicants have discovered that the above enzymatic compositions can be obtained and purified by a variety of methods. Medullary thyroid carcinoma tissue derived from rat, its cell lines, and / or cell culture media derived from these cells has been found to be particularly desirable sources of the crude alpha amidating enzyme. The crude alpha amidation enzyme can be purified by subjecting the crude composition to both size exclusion chromatography and anion exchange chromatography, especially strong. 20 for anion exchange chromatography. Herein, "strong" anion exchange chromatography refers to anion exchange chromatography performed on a resin that maintains a constant total positive charge in the pH range of 2 to 12. In certain preferred embodiments of the invention, size exclusion chromatography precedes strong anion exchange chromatography. and the size exclusion chromatography step may be preceded by yet another anion exchange chromatography. ·: ·. 25 graph steps. It may be desirable to carry out the second step of the anion exchange chromatography after the first step and, in a preferred embodiment, either the first one; | strong anion exchange chromatography or another strong anion exchange chromatography is performed at a basic pH, while the other is carried out at acidic pH.

I I t • t • · · • · · ·. · · ·. By using enzyme species that have been substantially purified to homogeneity, there is a *. enzyme specific monoclonal and polyclonal antibodies. purified enzyme as an antigen to induce an immune response in mice and chickens, respectively. In this way, antibodies raised from one species can be purified and π 110433 immobilized on a solid support to produce an immunoaffinity column specific for the enzyme. This column can be used to purify the crude enzymatic agent resulting in increased enzyme efficiency and reusability.

5

The alpha amidation enzyme, together with its tryptic fragments, has been sequenced by known methods and sequence information has been used to prepare oligonucleotide probes. Using labeled oligonucleotide probes prepared in this manner, the Applicants have isolated the gene encoding the alpha amidation enzyme from a cDNA protein library synthesized from polyA RNA derived from rat medullary thyroid carcinoma tissue. The gene, which is described in more detail in the detailed description of this application, may be linked to a suitable expression vector and transferred to a host cell capable of expressing the gene. Suitable hosts include e.g. E. coli. yeast strains such as S. cerevisiae or higher eukaryotic cells such as the cell line from which the enzyme was originally purified. Commercial mass production can be facilitated by a microorganism genetically engineered as described above to express large amounts of the alpha amidating enzyme.

Commercial mass production of enzymes from natural sources can be facilitated. 20 by means of separation methods which include both size exclusion chromatography and anionization. 'Exchange chromatography, whereby species retained by anion exchange chromatography are eluted. ''. using saline in a concentration greater than about 250 mM, and in particular 350 mM or; , ·. 500 mM. At high saline concentrations, most of the retained enzyme is eluted. ·. species. Size exclusion chromatography should be designed to isolate species with. '·. The apparent molecular weight is from about 58,000 to about 67,000 daltons, and in particular from about 60,000 to about 65,000 daltons. The purified preparation is capable of amidating a peptidyl compound which has been purified from natural sources or produced by recombinant DNA technology, i. L- • · • '': peptides consisting of amino acids.

• · * * · * • ·. · · ·. 30 In the figures • · * · ·. Λ images I to V refer to Example 1 and will be explained in greater detail later, • · '. '. Fig. VI to XII refer to Example 2 and will be explained in greater detail later, XIII to XVI relate to Example 2 and will be described in more detail below, 12,110,433. XVII-XXI relate to Example 4 and will be explained in more detail later, FIG. XXII relates to Example 10 and will be further described herein, and FIG. XXIII relates to Example 6 and is explained in more detail herein.

It has now been found that a homogeneous alpha amidating enzyme can be obtained by a multistep method using size exclusion and ion exchange chromatography on solid tumor tissue extracts, tumor cell lines, and tissue culture media derived from such cell lines.

The enzyme has been extracted from rat medullary thyroid carcinomas ("MTCs") developed in WAG / Rij Wistar rats as described by Roos, B.A., et al., Endocrinology, 1979, Vol. 150, # 1, pp. 27-32. This tissue is deposited under the designation IVI-10028 (subsequently ATCC 75168). The enzyme has also been extracted from other sources, in particular human and rat medullary thyroid carcinoma cell lines. Rat cell line 15 ("CA-77") was derived from rat medullary thyroid carcinoma tumors in serial production as disclosed by Muszynski, M. et al., JBC 1983, Vol. 258, pp. 11678-83. This cell line is deposited under the designation IVI-10029 (subsequently ATCC CRL 10919).

The human cell line ("HTT 54 (34)") was developed by B.A. Roos, VA Medical Center, Cleveland, Ohio, using human medullary thyroid carcinoma cells for primary culture. The human cell line HTT 54 (34) is deposited under the designation IVI-10031 (subsequently V min ATCC CRL 10918) (see "Purpose of Patent Procedure"). , where these deposits appear, and "Budapest Notificati-:. ·, is" No. 34, November 3, 1983, which are incorporated herein by reference in their entirety.

Specified tissue culture media from both human and rat cell lines have been found to contain significant levels of alpha-amidating enzyme activity: '1.! indicating that part of the enzyme is secreted from the cells. The enzyme can be prepared and purified, in particular by first subjecting the crude material (which contains the consumed culture media) to the anion-exchange chromatography (dyes, enzyme, and impurities). The sample can be: ''; 30 for example, in bulk to a preparative anion exchanger such as diethylaminoethyl: · 1>. ("DEAE") for a cartridge such as the CUNO 250 manufactured by CUNO Corp.

· · 13 110433

The composition eluted from the cartridge containing the alpha-amidating activity is then subjected to size exclusion chromatography on a resin of suitable resolution, for example, a very fine Sephacryl S-200 manufactured by Pharmacia Fine Chemicals.

The activity-containing eluent fraction is then subjected to ion exchange chromatography using a strong anion exchange matrix. The resin that can be used is a strong Mono Q HR5 / 5 anion exchange resin made by Pharmacia Fine Chemicals and one or more column passages may be required to purify the enzyme to homogeneous. The Mono Q HR5 / 5 column has a particle size of 10 µm, has a cavity volume of 40%, and is a gel having a charged group of CH2-N * (0¾ µ) and an ionic capacity of 0.28-0.36 mmoles / ml.

Each purification step can be monitored for both protein content and alpha-amidation activity level. This information is used to calculate the specific activity of the enzyme, which serves as an indicator of the relative purity of the enzyme.

The molecular weight of peptideyl glycine alpha amidating monoxygenase purified according to the invention (deposited in rat-derived enzyme, IVI-10032, then ATCC 75145; 20 deposited human-derived enzyme, IVI-10033, then ATCC 75146) is n 60,000 to 65,000 Daltons as determined by gel filtration.

• · • · · •; . ·, The enzyme has been purified so that its specific enzyme activity is at least n.

• · · • · ·: ·. ·. 25 mU / mg protein and especially at least about 50 mU / mg protein. Particularly specific. · '. The activity is greater than about 150 mU / mg protein. The alpha amidating enzyme is also purified to have a single homogeneous well-defined band after electrophoresis on sodium dodecyl sulfate / polyacrylamide gels (SDS-PAGE).

. Purified peptidyl-glycine-alpha-amidating monooxygenase is used to amidate the alpha-carboxyl group of the terminal glycine-containing polypeptide where glycine acts as an amino-group donor. The substrate peptide or polypeptide can be: Purify from natural sources, synthesize it from amino acid components, or synthesize it by recombinant DNA technology The glycine-terminated polypeptide is combined with oxygen in the presence of an effective amount of an enzyme, and the amount of enzyme required is dependent on a number of variables well known in the art. : specific activity of the particular enzyme preparation, the amount and chemical nature of the substrate to be converted, the time at which the transformation occurs, and the temperature and pH of the reaction mixture.

Other variables known to those skilled in the art may affect the exact amount of enzyme required in a given situation. Oxygen is usually present in a molar excess in the reaction with respect to substrate concentration. The desired concentration of copper (II) ions can be provided with a copper salt whose anion does not already adversely affect the reaction. With an enzyme having a specific enzymatic activity of only about 1 mU / mg protein, maximum alpha-amidation occurs at a relatively high concentration of copper (II) ions of about 4 μΜ. As the purity of the enzyme increases, the concentration requirements for exogenous copper (II) ion are reduced. Enzymatic activity may also be increased by the presence of ascorbate ions, which may be provided by an ascorbic acid salt, as long as the reaction is not adversely affected by the salt cation. For the purified enzyme, which has a specific enzymatic activity of about 50 mU / mg protein, maximal alpha-amidation activity occurs in about 5 mM ascorbate. The alpha amidation activity can be increased by the addition of catalase. The optimum pH for converting the biologically relevant substrate 20 to amidated products is 6.5 to 7.5.

Monoclonal and polyclonal antibodies directed against the enzyme are available. Using a homogeneous enzyme as an antigen to produce an immune reaction

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• · '· in mice and chickens. Applicants have prepared both monoclonal and polyclonal. ·: 25 antibodies as shown in Example 8. Stocks of specific antibodies for the alpha amidation enzyme are maintained in the applicant's laboratories.

1: Antibodies may be immobilized on a solid matrix which is insoluble in the media in which it is used. In particular, the matrix is one which is resistant to degradation.

. · ·. The matrix is provided with a functional group capable of binding proteins, such that,. '. the functional groups are then covalently bound to the antibodies. Such immobilization of antibodies '· t' facilitates the isolation of the α-amidating enzyme from natural and / or recombinant sources. This is accomplished by mixing immobilized antibodies with crude preparations of enzyme 110433. Antibodies specifically bind α-amidating enzyme molecules. Contaminating proteins do not bind to antibodies and are readily removed by gentle elution or gentle centrifugation. After removal of the impurities, the α-amidation enzyme can be removed from the immobilized antibodies by changes in ionic strength 5 or pH or by addition of chaotropic ions (Affinity Chromatography: Principles and Methods, Manual, Pharmacia Fine Chemicals, Uppsala, Sweden) and recovered in high purity.

The enzyme has also been sufficiently purified to determine its amino acid sequence. io This information was used to isolate the nucleic acid coding of the enzyme.

Thereafter, coupling to a suitable single-cell organism or host cell isolated from the multicellular organism is performed by standard recombinant DNA techniques (see Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold 15 Spring Harbor, 1982, or Wu, R.). , Methods in Enzymology, Vol. 68, Academic Press, 1979, which are incorporated herein by reference. The resulting cells, which contain heterologous DNA coding for the alpha amidation enzyme, allow sufficient enzyme production to be carried out in vitro for post-translational alpha amidation, and theoretically these cells allow this variant of the peptide or polypeptide to be performed in vivo.

• ·. ·. Although the present invention has been described in connection with its preferred embodiments, the art:. *. many variations and modifications will be apparent to those skilled in the art. Preferred embodiments of the invention are further explained by the following examples.

• ·: T: 25

EXAMPLE 1 Purification Method for the Alpha-Amidomalt Enzyme Prepared from MTC Tumors Frozen rat MTC tumors were ground to very small fragments and homogenized in 150 mM Tris pH 7.5 containing 250 mM sucrose, 0.25% N-acetyl-glucopyranoside, 0.1 mM PMSF, 20 pg / ml pepstatin and 100 pg / ml soybean trypsin inhibitor. Conventionally, 25 g of tumor tissue was homogenized in 200 ml of the above 16,104,333 buffered buffers on ice. Homogenization was accomplished by four 20-second crushes, each using a Polytron homogenizer (Brinkman) at setting 7. The homogenate was centrifuged for 15 minutes in a 9.000 g JA-20 rotor (Beckman) and the supernatant (Sup. 1) decanted. The pellet was re-homogenized in 60 ml of homo-5 genotyping buffer for three 20 seconds crushing each time at 7. This second homogenate was also centrifuged for 15 minutes at 9,000 g. The supernatant (Sup.2) of this centrifuge was combined with Sup.l. The combined Sup.l and Sup.2 were then centrifuged for 30 minutes in a 100,000g SW28 rotor (Beckman) at 4 ° C. To the clarified homogenate (Sup.3) sufficient ammonium sulphate phase crystals were added to form a 25% solution in ammonium sulphate. The crystals were added gradually over 15 minutes with homogeneous stirring at 4 ° C. After further stirring for 30 minutes at 4 ° C, the mixture was centrifuged for 15 minutes in a 27,000 g JA-20 rotor (Beckman) at 4 ° C. The supernatant was decanted and additional ammonium sulfate was added to the supernatant to make the solution in 40% ammonium sulfate. After further stirring for 30 minutes, the mixture was further centrifuged for 15 minutes at 27.000 g as described above. The supernatant of this spinning was discarded and the pellet containing at least 50% of the total enzyme activity of the homogenate was resuspended in 50 mM Tris HCl, pH 7.0 to form a sample. The activity was 8.2 mU / mg protein.

20 '".' Seohacrl S-300 Gel Filtration Exclusion Chromatography Sephacryl S-300 (Pharmacia) was equilibrated in 50 mM Tris HCl pH 7.0 and poured into. K 50/100 column (Pharmacia) according to the manufacturer's instructions The column volume was about 1.3 liters The sample was loaded onto the column in an amount of about 3% of the column volume. Proteins were eluted from the column in 50 mM Tris HCl at pH 7.0 at a flow rate of about 2 ml / min, 10 ml fractions were collected and analyzed for alpha-1: amidation enzyme activity using dansyl-D-Tyr The enzyme eluted from the column as a single peak of activity, as shown in Fig. I at a molecular weight of 60,000 to 65,000 daltons. Activity was; · At least 50 mU / mg protein.

»* I

i k I

• · 17 110433

Mono O Chromatography at pH 6.0 - Strong anion exchange chromatography

Fractions of the Sephacryl S-300 column containing maximal enzyme activity were pooled and dialyzed against 20 mM bis-Tris buffer, pH 6.0.4 liters. Liquid 5 was then loaded onto a Mono Q HR 5/5 column (Pharmacia) pretreated according to the manufacturer's instructions and equilibrated in 20 mM bis-Tris pH 6.0. The non-column bound proteins were collected in the column eluent (flow-through) and then the bound proteins were eluted with a linear salt gradient of 0-300 mM NaCl in 20 mM bisTris, pH 6.0. The column was run at a flow rate of 0.5 ml / min. and in each case 2 ml fractions collected? added. The protein solution eluted from the Mono Q column at pH 6.0 was immediately neutralized by collecting in tubes containing 200 μΐ of 1.0 M Tris pH 7.0 each. Fractions were analyzed for alpha amidation enzyme activity as above and a peak of activity eluted from the column in about 160 mM NaCl was observed (Fig. II). The activity was 161 mU / mg protein.

15

Mono O chromatography at pH 8.0

Fractions containing the peak of alpha-amidating enzyme activity from Mono Q chromatography at pH 6.0 were dialyzed against two 4 L shifts, of which The 20 peaks contained 50 mM Tris HCl pH 8.0. The enzyme was then loaded onto a Mono Q HR 5/5 · 5 'column (Pharmacia) equilibrated with 50 mM Tris HCl pH 8.0. Proteins 1 µl were eluted with a salt gradient of 0-300 mM HCl in 50 mM In Tris HC, pH 8.0, flow rate was 0.5 ml / min and 2 ml fractions were collected, each fraction was neutralized by the addition of 200 μΐ 1 , 0 M Tris pH 7.0 for each manifold.

. ·: ·; 25 lists the peak of enzyme activity by elution in 190 mM NaCl and 220 mM

In NaCl (Fig. III). The activities were at least 80 mU / mg protein and resp. 400: ': mU / mg protein.

• · t t »• · ·

Mono O-chromatography. pH 8.0. gel fraction electrophoresis of peak fraction of enzyme. '· 30% · «k

An aliquot of the peak enzyme fraction eluted in 220 mM NaCl and 408 mU / mg protein was applied to a 10% SDS-polyacrylamide gel after being heat denatured in buffer containing SDS. and B-18 110433 mercaptoethanol (Fig. IV). A single band was observed from the fraction eluted in 220 mM NaCl. The apparent molecular weight of this band is about 73,000 to 75,000 daltons.

Reinforcement of the band identity of 73,000-75,000 Daltons as alpha amidation enzymes 5

Aliquots of the excess alpha-amidation enzyme preparation purified using the above criteria were used for electrophoresis on a non-denatured 10% acrylamide gel. 50 μ!: Η sections were placed in each lane. After electrophoresis, the second band protein was visualized using the silver staining method. 3 mm strips were cut from the second strip and each strip was incubated in one well of a microtiter plate. 100 μΐ of a mixture of dansyl substrate, catalase, ascorbic acid and 150 mM Tris pH 7.0 was added to each well. Incubate for 16 hours at 37 ° C with continuous shaking. Quantitative portions of each well were then analyzed for conversion to substrate amidated product. Enzymatic activity was observed in two gel slices. The activity migrated along the heavily stained protein band in the corresponding stained gel band (Fig. V).

Example 2 20 "V Methods for Preparing Recombinant Calcitonin of Human Origin Human Calcitonin is a 32-amino acid peptide hormone having an amidated prolyl residue at its carboxyl terminus. The microorganism was genetically manipulated by a recombinant. - • · · · · · · · to form 25 fusion proteins containing the amino acid sequence corresponding to human calcitonin. The fusion protein gene was designed with the human calcitonin: 1 ·, quentum backed by an arginyl residue at its amino terminus and its carboxyl terminus. '': with a wart residue that also terminated the recombinant fusion protein (see Fig. VII). After incorporation of the human t «», ·, · calcitonin gene into a plasmid to form a suitable fusion plasmid, the microorganism was transformed with this plasmid and obtained. fusion il .. May 1st (see Fig. VI) This is human calcitone ini protein was isolated by recombinant

»I

'. t 1; from lysates of nant microorganisms by precipitation. The cysteine residues were converted to S- · sulfonates and the lysyl residues were reversibly blocked by reaction with citric acid anhydride 19 110433. Since human calcitonin does not contain arginine, tryptic digestion of the recombinant fusion protein yielded a peptide containing the human calcitonin sequence with a carboxyl terminal glycine extension. This peptide was isolated by either reverse phase HPLC (see Figure VIII) or ion exchange chromatography, and its structure was confirmed by amino acid and micro sequence analysis.

The glycyl residue of the peptide was removed and the last-last prolyl was converted to prolinamide with the alpha-amidating enzyme of the present invention. An example of the semi-preparative alpha-amidation conditions of this peptide is as follows: Lyophilized pep-10 (200-300 nanomolar) (substrate) was dissolved in 200 µl of 150 mM Tris-HCl buffer, pH 7 containing about 750 µL of alpha -amidointientsyymivalmistetta. The enzyme was derived from either a rat MTC tissue or a spent tissue culture medium from a rat MTC CA-77 cell line. The enzyme was purified to the extent that all proteolytic activity was removed. This was accomplished by a combination of ion exchange and exclusion chromatography (see method above). Pure, homogeneous enzyme was not a requirement. To this mixture was then added ascorbic acid and copper sulphate in sufficient amounts to give final concentrations of about 3 mM and respectively. 2 µM. The resulting solution was mixed and incubated at 37 ° C for 5-6 hours. Conventionally, the percentage of conversion to a substrate product is 70 to 90%. S-. After removal of the sulfonate groups, the recombinant human recombinant calcitonin (rhCT) «·" V was purified by reverse phase HPLC (see Fig. IX). The final product was characterized by its retention by reversed phase HPLC (see Fig. X), quantitative tryptic mapping, ·, (see Fig. XI), amino acid analysis (see Table I) and its biological activity (see Fig. XII). recombinant human calcitonin • ·. 1: ·; 25 could not be separated from human synthetic calcitonin.

: 1 ·, I The above shows that the preparations obtained according to the present invention are capable of amidating a physiologically relevant substrate, e.g., human calcitonin, which is · · ·, ·. ·, Produced by recombinant DNA technology, m.p. contains only L-amino acids.

• ·. '' 1. 30> · • »a I> ·.

> »I

• · · t> I • »I • 4 20 110433

Reference Examples

Comparison of Activity of the Prescribed Preparation with Prior Art Comparison of Analytical Systems to Determine Specific Activity of the Prepared Preparations

Several activity analysis systems have been used in the prior art. Most of the works known in the art have used analysis based on the conversion of DTyr-Val-Gly to D-Tyr-Val amide. This assay uses a radiolabeled tracer (I-DTyr-Val-Gly) mixed with an excess of unlabeled material (DTyr-Val-Gly). Measuring the conversion of a labeled tracer allows extrapolation to unlabeled material, and this in turn allows for the calculation of activity.

Although Applicants have used this assay, assays for the activity of the disclosed preparations are based on direct measurement of the conversion of dansyl-Tyr-Val-Gly to dansyl-Tγ-Val-amide.

To allow meaningful comparison of the specific activity of known preparations. Experiments have been conducted to compare analytical systems to the formulations of this invention.

• · · • · * I · • · ♦ ·; The trial protocols were as follows: ». 25 I. Monodanswli-L-Tvr-Val-Glv • · · - - •; | In these experiments, an alpha-amidating enzyme preparation was used as the enzyme source, isolated from rat medullary thyroid carcinoma tumors and tissue culture medium. v, those harvested from CA-77 cells. The concentration of enzyme used was in all. · · ·. 30 experiments unless otherwise noted.

»·» * »» »» T • * »* · 21 110433

The reaction mixture for the conversion of the monodansyl substituent contained: 5 μΐ enzyme 5 μΐ 30 mM ascorbate 5 5 μΐ 20 μΜ CuSO4 5 μΐ 100 pg / ml bovine pancreatic catalase 5 μΐ containing 2 nmole substrate 25 μΐ 150 mM TES pH 7.0 10 Samples were prepared in duplicate they were incubated at 37 ° C for 10.20 and 30 minutes. The enzymatic reaction was stopped by the addition of 10 μΐ 500 mM EDTA. The substrate and product were separated by reverse phase HPLC using a Hewlett Packard-1090 liquid chromatography system and assayed using an HP-3392 integrator. The conversion of monodansyl-L-Tyr-Val-Gly to the alpha-amidated product is shown to be linear over time.

II. 125I-D-Tvr-Val-Glv D-Tyr-Val-Gly and D-Tyr-Val-NH 2 were iodinated with iodine balls of Pierce Chemical Company.

. The radiolabeled substrate and product were used to calibrate the sulfylpropyl * cation exchange column. 125I-D-Tyr-Val-Gly was added to 650 pMD-Tyr. Val-Gly and used as a substrate. Reaction Mixture Monodansyl Substrate Mod. for msex contained: • · · 14 · · »* · I · • ·, 'j1. 5 μΐ enzyme 5 μΐ ascorbate (30 mM): '·,: 5 μΐ 100 pg / ml catalase 5 μΐ20 pMCuSO ^, ·, *, 5 μΐ substrate, 650 μΜ final conc.

. "1. 30 25 μΐ 150 mM TES pH 7.0 ki ki» »Näyt Samples were incubated for 10.20 and 30 minutes at 37 ° C. The reaction was stopped by the addition of 500 mM KES. EDTA: The whole sample was diluted with 10 mM sodium phosphate buffer, 22 110433 pH 5.2, and applied to a sulfyl-propyl-cation exchange column The substrate does not bind to the column The amidated product was eluted with 500 mM NaCl.

5 III. The reaction conditions used for the amidation of D-Tvr-Val-Glv D-Tyr-Val-Gly were the same as for dansyl and iodinated substrates. The reaction mixture had a substrate concentration of 650 µM. The separation of the substrate and the product is accomplished by gradient elution by reverse phase HPLC using an HP-1090 liquid chromatography system. The column effluents were monitored at 280 nm. The sensitivity of this assay is much lower than that of dansyl or iodinated substrates. To accommodate this lower level of sensitivity, alpha-amidation reactions were conducted over longer periods of time and / or with higher amounts of alpha-amidation enzyme.

analysis of the split values of 125I-D-Tyr-Val-Gly to 125I-D-Tyr-Val-amide and dansylTyr-Val-Gly to dansyl-Tyr-Val-amide shows that about 1 is converted at each time point, 55 times more iodinated substrate than dansyl substrate. Thus, a conversion factor of 1.5 times the activity determined by the Danesian substrate method (s) must be used when comparing the prior art analytical system to the analytical system used by applicants.

• i • i I »· · • ι i ·,! * Some more stringent kinetic analysis has also been used (by applicants) »». for comparison of dansyl Tyr-Val-Gly analysis (known in the art) to D-Tyr-Val-Gly- • · ·; ·. ·, For analysis. This analysis shows: »jc * · · I I I i

Substrate Km Vmax D-Tyr-Val-Gly 37 31 • * (Prior Art) i · »Dansyl-Tyr-Val-Gly 1.7 21 • 1 I • ·, · **, 30 (Applicants) • · II» I> »* * *

I I

As can be seen by comparing the maximum velocity of these two substrates • i * (Vmax = pmol product / min / μΐ) D-TyrVal-Gly (state of the art) produces about 1.48 as much activity as dansyl Tyr -Val-Gly (applicants). This is consistent with and confirms the above findings.

Comparison of activity

Eipper et al. (PNAS) on page 5147, fig. 4 that Vmax is 39 picomoles / microgram / hour. This corresponds to a specific activity of 0.65 mU / mg protein / min. This is the highest activity so far reported in the art. By dividing this by the above conversion factor 1.5, a specific activity of 0.4 to 10 mU / mg protein / min is derived. This value can now be directly compared to the specific activities mentioned above achieved by the applicants. Applicants, as discussed above, have achieved activities of at least 25 mU / mg protein and greater than 1500 mU / mg protein. Applicants have thus achieved an activity of 60 - more than 3,750 times that of Eipper (PNAS).

15

Example 3

Purification and Characterization of Rat MTC Tumor Alpha Amidation Enzymes 20. '* *: Frozen rat MTC tissue was ground to very small fragments and homogenized. · In aqueous buffer using a Polytron homogenizer. At low speed •. ·; After centrifugation on the supernatant, the supernatant was saved and the pellet re-extracted: *. · Fresh buffer. This second homogenate was again subjected to low speed centrifugation: * 25 and this supernatant was combined with the first. The two combined supernatants were then clarified by high speed centrifugation and the supernatant of this centrifuge was used as a starting material to purify the enzyme.

The high-speed centrifuged supernatant was subjected to ammonium sulfate fractionation.

'·. · * 30 Most of the enzyme activity was found to be precipitated with 26-40% ammonium | sulfate fraction and the pellet of this fraction was further purified as shown below.

• · · »· 24 110433

Exclusion chromatography was performed on a Sephacryl S-300 column. In Example 1, the entire enzyme was eluted from this column at a single peak of activity. In this example, the column length was increased and the column flow rate decreased. Under these new elution conditions, a larger peak of activity was seen (as in Example 5 1), followed by a smaller downstream peak, which may correspond to a lower molecular weight form of the enzyme. At this point, it is unclear whether the low molecular weight form of the enzyme occurs in vivo or is produced by partial proteolytic digestion during extraction and purification.

The larger peak of the S-300 column activity was pooled and chromatographed on a Mono Q column at pH 6.0. In this example, a larger, preparative column was used than in Example 1 (Mono Q HR 10/10) eluted using a less steep linear salt gradient. As a result of these changes, four peaks of alpha amidating enzyme activity were detected at this stage, eluted in 160 mM, 200 15 mM, 220 mM and 240 mM NaCl (peaks I, H, III and IV of Figure XIII). This indicates that there are various forms of the enzyme and that these forms have charge heterogeneity. Further, the polyacrylamide gel analysis of the proteins at the enzyme activity peaks shows that peaks II, III and IV contain an alpha amidating enzyme of approximately the same molecular weight (i.e., 73,000 to 75,000), whereas peak I: *: 20 contains a different, possibly lower molecular weight. Hui- IM ·. The purine activity was purified to homogeneity as follows: Peak III enzyme was pooled and chromatographed on a Mono Q HR 10/10 column pH- *. 'At 8.0 (Fig. XIV). The enzyme was eluted from this column as a single peak in 250 mM NaCl and gel analysis confirmed that the enzyme had been purified to homogeneity (Fig. XVa, lane 6). The following characterization tests were performed on Peak III purification results.

• I I

The molecular weight of the peak III enzyme was determined to be about 75,000 daltons by 7% polyacrylamide gel analysis (Fig. XVb).

: *. 2. The pH optimum for enzyme activity was determined to be 5.0-5.5 using N-dansyl-Tyr-ValGly as a substrate (Fig. XVI). However, due to the increased stability of the enzyme at 110433 neutral pH, it may be advantageous to carry out an amidation reaction at such pH.

3. It was determined that the amount of copper required as the cofactor for enzyme activity was inversely proportional to the purity of the enzyme. The homogenized purified enzyme required 0.1 μΜ or less Cuu ions for maximal activity, while the crude enzyme preparations required 2 μΜ Cu ^ ions.

4. The isoelectric point (pI) of the enzyme was 4.8.

5. The specific activity of the peak III homogeneous enzyme was 2,100 mU / mg protein.

Peak II enzyme was also purified to homogeneity. However, with this enzyme, it was found that Mono Q chromatography at pH 8.0 was insufficient to prepare a homogeneous preparation. Therefore, the entire enzyme (Fig. XIII) of the Mono Q column, pH 6.0, peak II was dialyzed against 1M Tris, pH 7.0, and loaded onto a phenyl sepharose column equilibrated with the same buffer. The innermost portion of the contaminating protein species was recovered by column flow and the amidating enzyme eluted at a later stage was substantially purified. Further purification of the combined enzyme on the Phenyl Sepharose column, '' on a Mono Q HR 10/10 column, pH 8.0, gave * «♦

*: A homogeneous preparation of peak II enzyme eluted from the column at 220 mM

• ·:.:: In or above NaCl.

Characterization of the peak II enzyme confirmed that *; * · 1. The molecular weight of the peak II enzyme at 7% polyacrylamide gel electrophoresis was about 73,000-75,000 daltons. Thus, peak I and A peak III enzymes were not present. could be distinguished • »v. * by comparing their molecular weights.

'··· * 30

| 'M' · 2. The pH optimum for peak II enzyme activity was 5.0-5.5. Again this peak II

:: The enzyme has the same property as peak III enzyme.

26 110433 f! 3. The isoelectric point (pi) of the peak II enzyme was about 5.8.

Example 4 Purification and Characterization of 5α-Amidation Enzyme Derived from CA-77 Cell Tissue Culture Media

The rat medullary thyroid carcinoma cell line, CA-77, was grown as a monolayer in 150 cm 3 T flasks (Corning) at 8% CO 2. The culture was maintained in the specified medium containing Dulbecco's Modified Eagle Medium: F-10 (1: 1), 3.7 g NaHCO3, 5 pg / ml transferrin, 10 pg / ml insulin, 30 nM selenium and 4 pg / ml gen tamycin sulfate. Cultures grown in this manner could be stored indefinitely if the medium was changed every 48 hours. To increase the cell strain, they were subcultured and grown for three days in medium containing serum (5% horse and 2.5% 15 fetal calf serum). The cells were then washed twice with phosphate buffered saline and supplemented with a prescribed medium.

Tissue culture media were harvested aseptically over a 48-hour schedule and stored at -20 until purification. Tissue culture media (conventional 6: *: 20 liters) was diluted with 2 liters deionized water (3: 1) and placed at 50 ml / min in a weak DEAE anion exchange cartridge (Cuno = f = 250) above. equilibrated with 1.0 L of 20 mM bis Tris: HCl, pH 6.0.4 ° C.

:. ·: Fa-AE ") was gradually eluted from the cartridge at a flow rate of about 50 ml / min. 50 mM

:. Tris HCl pH 7.0 containing 500 mM NaCl. Fractions of two anion exchange preparations * · · * · 'containing alpha-AE activity (specific activity 10-15 mU / mg) were pooled and concentrated 4 to 5-fold under reduced pressure using Savant t · * RH-100 prep. rotor.

«· *. v This material was applied directly to a 5 x 50 cm column containing Sephacryl 300-> · · · 30 SF (Pharmacia). The mobile phase was 100 mM Tris: HCl, pH 7.0.1.0 ml / min. at a flow rate. All gel filtration chromatography was also performed at 4 ° C (Fig. XVII).

• · 27 110433

The amidation enzyme preparation at this purification step is free from non-specific proteolytic activity and has an activity of at least 50 mU / mg protein. The amidation enzyme preparation of this step has been used successfully to amidate recombinant Gly-expanded human calcitonin and growth hormone releasing factor.

Starting at a cell density of 1 -1.5 x 10 6 cells / ml (from T-flasks), we have conventionally obtained a yield of 200-350 mU of amidating enzyme activity / liter of medium after these two purification steps. The enzyme is stable and suitable for use as a solution or after immobilization on a solid support.

The column fractions containing alpha-AE activity were pooled and then dialyzed against 20 mM bis Tris: HCl, pH 6.0.6 L. The enzyme was loaded on a Mono Q HR 10/10 strong anion exchange column (Pharmacia) previously equilibrated with 20 mM bis Tris: HCl, pH 6.0. The enzyme was eluted from the column using a linear gradient of 0-300 mM NaCl over three hours at a flow rate of 2.5 mL / min. Four chromatographically different forms of alpha amidating enzyme activity were separated in this purification step. The peaks were numbered in the column elution estimate (Fig. XVIII). Peaks III and IV represent the higher molecular weight forms of the enzyme and correspond to peaks derived from MTC tumor Ila and III. Peaks I and II denote lower molecular weight forms of the enzyme, which may be proteolytic fragments of peak III: IV and IV.

• '.: The four different forms of alpha-amidating enzyme identified by our laboratory • •:. · Differ in their total surface charge, as indicated by various retention times during strong anion-exchange chromatography (Fig. XVIII). These · · t

The pH optimum of four chromatographically different forms of V * 25 is also different. XIX

the results show that at the peaks the lily IV has a similar pH optimum of 5.0 to 5.5. These results are consistent with the pH optimum determined for peaks II and III purified from the MTC tumor. Peak I and II have a much wider pH activity range which:.:. * is 5 to 8.5 (Fig. XIX). These results are in close agreement with the pH optima reported by Eipper et al., Peptides, Vol. 4, pp. 921-28 (1983) and Murthy et al., J. Biol.

Chem., Vol. 261, pp. 1815-22 (1986).

• »,» · • · · · · 28 110433 ΙΛί

The radioactive labeling of the enzyme from peaks II and IV by Na I and SDS-PAGE confirmed that the peak Mole molecular weight of the enzyme IV was 73-75 kDal, whereas the peak II had less than 55 kD. The exact molecular weight is unknown for Peak II because it was not purified to homogeneous (several protein bands are apparent in the 45-55 kDal range).

Peak III and IV enzyme can be purified to homogeneity using a combination of hydrophobic interaction chromatography and strong anion exchange chromatography at pH 8.0. The activity of Peak IV enzyme (Fig. XVIII) was pooled, concentrated to about 10 ml in vacuo and directly applied to a 1.3 x 8 cm column containing phenyl sepharose (Pharmacia) equilibrated with 500 mM Tris: HCl. at pH 7.0. Fractions containing alpha-AE activity were eluted with equilibration buffer at a flow rate of 0.5 ml / min. (Fig. XX). Peak fractions containing alpha amidating activity were pooled, dialyzed against 50 mM Tris: HCl, pH 8.0, then loaded onto a Mono Q HR is 10/10 column equilibrated with 50 mM Tris: HCl, pH 8 , 0. The enzyme was eluted from the column using a linear gradient of 0-300 mM NaCl over three hours at a flow rate of 2.0 mL / min. (Fig. XXI). Fractions containing alpha-AE activity eluted at 240 mM or greater than about 240 mM were pooled, adjusted to 0.001% (v / v) in Triton X-100 and stored at 4 ° C. The specific activity of the purified enzyme: ': 20 was determined to be about 1500 mU / mg protein at pH 7.0. Hui-. ' · ·: Pur III alpha-AE activity was purified to homogeneity using peak IV coupling. "/ · D.

• I

• · · • · · * • · ·: *. · Physico-chemical properties of tumor peak (eg 3) and tissue culture peak (eg 4): • 25 characteristics including molecular weight (73,000-75,000 Daltons), pH optimum (5.0-5.5), amino terminal sequence and elution position (greater than about 240 mM sodium • ·

t t I

'·' · Chloride from strong anion exchange chromatography, carried out at pH 8.0), indicates that the two peaks can denote the same enzyme.

• ·

• «I

• · · • · * • t * I * I »·

Example 5 29 110433

Alpha-amidation of biologically relevant peptide hormones using alpha-amidation enzyme 5

Several recombinant peptide hormone substrates, including salmon and human calcitonin, human growth hormone releasing factor and human calcitonin gene related peptide substrates, have been successfully prepared by the alpha-amidating enzyme preparation of this invention. The methods used to prepare recombinant salmon calcitonin, a human calcitonin gene related peptide, and human growth hormone releasing factor are shown below as an example. Similar methods can be used for other recombined peptides.

Salmon calcitonin is a 32-membered peptide hormone having an alpha-amidated prolyl residue at its carboxyl terminus. The microorganism was manipulated to produce a genetically recombined fusion protein containing the salmon calcitonin corresponding amino acid sequence. The fusion protein gene was constructed by combining the salmon calcitonin sequence with a methionyl residue at its amino terminus and at its carboxyl terminus with a gly-: 20-yl residue, which also terminated the recombinant fusion protein. After the salmon calcitonin • · / · · * gene was inserted into the plasmid, the microorganism was transformed with this plasmid and expression of the fusion protein was achieved. This calcitonin-containing protein was isolated from the lysates of recombinant microorganism by precipitation and its cysteinyl residues were converted to i.s .: S-sulfonates. Since salmon calcitonin does not contain methionine, the cyanobromide cleavage of the recombinant fusion protein yielded a peptide containing the salmon calcitonin sequence extended by carboxyl-terminal glycine. This peptide was isolated by either reverse phase HPLC or ion exchange chromatography and its structure; 'Was confirmed by amino acid composition and micro-sequence analysis.

The last of the last prolyl residue was converted to proline amide by the action of the alpha amidation enzyme. An example of the conditions used for alpha-amidation of this peptide is as follows: Lyophilized peptide substrate (glycine-extended salmon calcitonin precursor, 200-300 nanomolar) was dissolved in 200 µl of 150 mM Tris-HCl buffer, pH 7.0, 110433 containing about 750 µU alpha-amidating enzyme. The enzyme can be derived from tissue culture media derived either from MTC tumor or rat MTC CA-77 cell line. The enzyme must be purified to such an extent that all foreign proteolytic enzyme activity is removed. This is usually accomplished by a combination of ion exchange and exclusion chromatography (see Example 4). A pure, homogeneous enzyme is not a requirement. To this mixture was then added ascorbic acid and copper sulfate in sufficient quantities to give final concentrations of about 3 mM and respectively. 2 μΜ. Catalase (7.5 pg / ml), ethanol (1%, v / v) and potassium iodide (50 mM) can also be added to the reaction mixture to improve the yield of alpha-amidated salmon calcitonin. The resulting solution was mixed and incubated at 37 ° C for 5-6 hours.

The S-sulfonate groups were then removed by treatment with beta-mercaptoethanol, after which the recombinant salmon calcitonin was purified by reverse phase HPLC. The final product was characterized by its retention by reverse phase HPLC, quantitative tryptic analysis, and amino acid analysis. In all cases, recombinant salmon calcitonin could not be distinguished from synthetic salmon calcitonin.

The human calcitonin gene-related peptide is a 37-membered peptide hormone with an alpha-amidated phenylalanyl residue at its carboxyl terminus. The fusion protein gene: ': 20 was constructed in the same way as salmon calcitonin (see above) such that human calc. the tonin gene related peptide sequence was joined by a methionyl residue at its amino-> · · • "/ · terminus and its carboxyl terminus by a glycyl residue which also terminated a recombinant human calcitonin gene-related peptide. i *: Diesel release, purification, alpha amidation and characterization were also performed as described above for t · ·: * 25 recombinant salmon calcitonin.

• ·

I i I

* · '' Human Growth Hormone Releasing Factor (hGHRF) is a 44-membered peptide hormone, · *; * * having an alpha-amidated leucyl residue at its carboxyl terminus. The hGHRF fusion protein gene was constructed by combining the amino acid sequence of the peptide hormone with a · · · · 30 tryptophan residue at its amino terminus and its carboxyl terminus with a glycyl residue which also terminated the recombinant fusion protein. fusion containing hGHRF »»:. The protein was isolated from the lysates of the recombinant microorganism by precipitation. The non-hGHRF portion of the fusion protein was denatured by converting cysteinyl residues to S-sulfonate derivatives. Since hGHRF does not contain tryptophan, chemical digestion of the recombinant fusion protein with BNPS-skatole reagent would oxidatively cleave the fusion protein to form unamidated hGHRF expanded with carboxyl-terminal glycine. Simultaneously, the methionyl residue at position 27 of the hGHRF molecule was oxidized to 5 methionine sulfoxide. This glycine-expanded peptide was isolated using gel filtration and reverse phase HPLC. Its structure was confirmed by amino acid analysis of trypsin digested fragment peptides.

The last previous leucyl residue was converted to leucine amide by the alpha amidating enzyme of this invention. An example of the preparation conditions for alpha-amidated hGHRF is as follows: Lyophilized peptide substrate (20-40 nanomolar) was dissolved in 150 µl deionized water and mixed with 90 µl (500 pU) (pH 7.0) alpha-amidating enzyme preparation derived from either rat Tissue culture media from MTC tumor or rat CA-77 cell line. The enzyme had to be purified to remove all foreign proteolytic enzyme activity by a combination of size exclusion and ion exchange chromatography. Then, ascorbic acid and copper sulfate were added to the enzyme / substrate mixture in sufficient quantities to give concentrations of 3 mM and respectively. 2 pM. The resulting solution was mixed and incubated at 37 ° C for 4-6 hours. The usual percentage conversion of substrate to product is 95% based on amino acid analysis of trypsin: thawed fragments. Finally, the methionine sulfoxide residue was reduced to methionine with 4 M beta-mercaptoethanol buffered at pH 4 with 10 mM sodium acetate at 80 ° C for one hour. Final • · i .:: The product was purified by reverse phase HPLC and characterized by its retention time, tryptic ·. * by digestion analysis and amino acid analysis. The recombinant alpha-amidated product was also tested for biological activity. In all cases, the recombinant hGHRF could not be separated from the synthetic hGHRF.

»* · I · · • · • I k

In addition to the above studies, two commercially available glycine-expanded '· * · * peptide hormones were evaluated for their ability to serve as substrates for alpha-' *; · * 30 for the amidation enzyme. These are precursors of the alpha-melanocyte-stimulating hormone and ': Substance P. In both cases, the results indicate that both peptides are »· *. '': Suitable substrates for the alpha amidating enzyme.

32 1 10433

Example 6

Sequence analysis of purified rat alpha amidation enzyme 5 Fractions containing purified alpha amidation enzyme were pooled from either rat MTC tumor tissue or CA-77 cell culture supernatants, and the sulfhydryl groups of the enzyme were subjected to reduction followed by carboxymethyl. The resulting reaction mixtures were then loaded onto a Vydac C4 reverse phase HPLC column (5 μΜ particle size, 33 nm pore size) equilibrated with 0.1% aqueous trifluoroacetic acid. The column was washed with this solution to remove excess buffer salts.

The desalted enzyme was removed from the HPLC column by elution with 80% acetonitrile containing 0.08% trifluoroacetic acid. The column effluent was monitored by UV at 220 nm. The resulting protein fractions were collected, pooled and lyophilized. This material was then redissolved in 100 µl of 0.1% SDS and then placed in a Applied Biosystems Model 470 A Protein Sequencer. The methods used in the micro-sequence analysis followed the instructions given by Applied Biosystems. The resulting phenylthiohydantoin amino acids were analyzed by HPLC on a Hypersil C18 column (particle size 5 M, pore size 10 nm) with absorbance monitored at 269 and 313 nm.

Hewlett-Packard 1090 liquid chromatography system. From the tumor tissue: the amino-terminal sequence of the peak (peak III) of the 20 major enzyme component was the following: · · · · 1 2 3 4 5 6 7 8 9 10 11 12 · · · · · · · · · · · · · · -Ser-Asn-Glu-Cys-Leu-Gly-Thr-Ile-Gly-Pro- \\ i 13 14 IS 16 17 18 19 20 21 22 23 24 25

Val-Thr-Pro-Leu-Asp-Ala-Ser-Asp-Phe-Ala-Leu-Asp-Ile-25 26 27 28

Arg-Met-Pro Head of enzyme (peak IV) from CA-77 cell tissue culture supernatants -:.:. Information on the amino-terminal sequence of the * component indicates that it is identical to * · * * '30 tumor tissue enzyme (peak III). However, a smaller component was also detected in the micro-sequence analysis of this enzyme, which appears to contain amino-. * · · Terminal extension compared to the major form of the alpha-amidating enzyme. The presence of this component is probably due to the different post-translational processing of the enzyme. The amino-terminal amino acid sequence of this component was as follows: 123 4 56789 10 11 12 NH 1 -Phe-Lys-Glu-Thr-Thr-Arg-Ser-Phe-Ser-Asn-Glu-Cys 5 13 14

Leu-Gly

Additional amino acid sequence values were obtained for α-amidating enzyme from rat MTC tumor tissue by the following procedure: Approximately 400 µg of purified enzyme was reduced and carboxymethylated. The enzyme solution was then transferred to a dialysis tubing and dialyzed against 25 mM Tris-HCl pH 8.0 / 0.5 M urea for 18 hours. The retentate was then transferred to a 1.5 mL centrifuge tube and concentrated to a volume of 600 µL under reduced pressure. To the enzyme solution was added 2 μΐ (2 pg) trypsin and the mixture was incubated for one hour at 37 ° C. At this point, a second aliquot of trypsin (2 µg) was added and incubation was continued for 2 hours at 37 ° C. Digitization was terminated by adding 200 µl of 4 M urea / 10% acetic acid. The extract was applied to a Vydac C18 reverse phase HPLC column (particle size 5 µm, pore size 33 nm) equilibrated with 0.1% aqueous trifluoroacetic acid. The column was then eluted with a linear gradient of acetonitrile to a concentration of 50% over 4 hours and fractions were collected over two minute periods. Reverse phase HPLC elution peaks for the tryptic extract of the enzyme are shown in Figure XXIII. The three tryptic peptides obtained were subjected to automated sequence analysis as above, and the results are shown below. (Peptides are labeled with their fraction number.) • * - * - Serp-peptide # 65 * - · - * - 25 -Ser-Met-Gln-Pro-Gly-Ser-Asp-Gln-Asn- His-Phe-Ser-Gln-Pro

Thr- I ««

* I I

• · · · · · · · · · · · · · · · · · · · Asn-Gly-Gln-Trp-Thr-Leu-Ile-Gly-Arg- · · · · · · · · · · · · · · · · · · · · · · · : o 86 :: -Phe-Val-Thr-Gln-Trp-Gly-Glu-

Example 7 34 110433

Molecular cloning of a gene or DNA sequence. encoding rat MTC or CA-77 cell line alpha amidation enzyme 5

The ability to clone a gene or DNA sequence encoding an alpha amidation enzyme is due to a number of critical pieces of information and reagents. A reliable source of the enzyme protein must be found, which in turn acts as the source of the enzyme messenger RNA (mRNA) and finally its complementary DNA (cDNA). Isolation of the enzyme gene or cDNA also requires a molecular probe of the enzyme in question. Generally, this molecular probe has one of two forms. It is either an oligonucleotide whose sequence is complementary to a portion of an enzyme gene, or is an antibody molecule (or collection of antibody molecules) that specifically recognizes an enzyme protein. The derivation of these molecular probes requires a method for purifying the enzyme such that either antibody specific to the enzyme can be prepared or the amino acid sequences of the enzyme can be determined to generate oligonucleotide probes. These conditions are satisfied for the alpha amidating enzyme of this application.

The alpha amidation enzyme was purified from rat MTC tissue and from media treated with rat CA-77 cells. This confirmed that these cell sources would contain the mRNA encoding the enzyme pro- * *: protein. The methods we have used to prepare the cDNA for the alpha-? · * Amidation enzyme are well known in the molecular biology field.

• * »t * •» f I <• t f '. Specific explanations of these methods can be found in laboratory manuals, such as

Molecular Cloning (1982), DNA Cloning, (Part 1) a Practical Approach (1985) or pri- '; * · For reference reference functionality such as Gubler, V. & Hoffman, B. J., Gene, Vol. 25, p.

262-69 (1983), or Young, R.A. and Davis, R.W., PNAS, Vol. 80, pp. 1194-98 (1983).

•. *. * These methods are generally applicable, with the critical variable being the only source of mRNA used. To prepare the alpha-amidating enzyme specific for cDNA, we used both rat MTC tissue mRNA and CA-77 cell :, · · mRNA. The double-stranded cDNA samples, which were synthesized, were gene libraries by well known methods.

35 110433

As discussed above, recognition of specific cDNAs for alpha amidating enzyme mRNA requires molecular probes that can distinguish these combinations from other combinations in a specific library. Examples 3 and 4 show in detail the purification methods used to prepare the enzyme required to conduct the molecular probes. Example 6 explains the use of this protein for the determination of amino acid sequences, while Example 8 explains the use of the purified protein for the preparation of enzyme-specific antibodies.

The amino acid sequences of Example 6 are sufficient for the preparation of specific selective oligonucleotide probes. In the preparation of oligonucleotide probes, several factors are important for making the probes selective. A complete disclosure of these can be found in Lathe, R. J., J. Mol. Biol., Vol. 183, pp. 1-12 (1985). Because generally more than one nucleotide sequence can encode the same amino acid sequence (this principle is known as the degree of degeneracy of the genetic code), a single nucleotide sequence represents only one of several possible gene sequences. To ensure that the gene in question is recognized by the oligonucleotide probe, an equimolar mixture of all possible nucleotide sequences that could encode an amino acid sequence specific for the amidation enzyme can be prepared. The complexity of such mixtures often renders them less than absolutely selective and thus generally requires more than one region of the amino acid sequence of the protein to be used in order to achieve absolute specific selectivity. · For a specific enzyme.

Alternatively, an oligonucleotide that is selective for the gene in question can be prepared by preparing a unique nucleotide sequence of sufficient length so that even a slightly imperfect complementarity to the desired gene produces a stable hyb- i · •; · 'Ridin. This unique sequence may have very little potential (length dependent · · *. *. *) To form a stable hybrid with other gene sequences. The unique * ... * 30 sequence is most often made up of a codon for each amino acid in the protein sequence. The frequency of use of a codon for particular species can be determined. Derives from the grouping of the corresponding amino acid sequences of known gene sequences and proteins of this species. These methods are well known to those skilled in the art of molecular biology.

Another way that can be used to prepare specific oligonucleotide probes is by attaching deoxyinosine residues to oligonucleotides at positions with maximum degeneration. This nucleotide substitution serves to reduce the degree of degeneration of the probe sample and thus may have beneficial effects on the selection process. (The use of deoxy inosine in an oligonucleotide probe is described in Ohtsuka et al., J. Biol. Chem., Vol. 260, p. 2605-08 (1985)).

10

We have used alpha-amidation enzyme protein sequences to design a number of oligonucleotide probes that can be used to isolate amidation enzyme cDNA. The sequences used and the probes we have prepared are shown in Table II. It should be noted that, if the molecular biologist had amino acid sequences, he could develop alternative methods to isolate the gene by probe hybridization.

The oligonucleotide probes thus produced have been used by well-established methods (see Molecular Cloning 1982) for screening both plasmid and bacteriophage cDNA libraries and for isolating the alpha amidating enzyme cDNA.

Table II

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Example 8 A. Generation of Monoclonal Antibodies to Alpha-Amidation Enzyme in Mice 511 Balb / cJ mice were immunized and boosted with purified amidation enzyme preparations. These mice were bled and serum treated and titrated against the buffered enzyme by ELIS A assay. The assay (techniques well established) was performed by absorbing the purified enzyme into a polystyrene plate which was then washed and blocked (to prevent non-essential binding of antibodies to the plate) with bovine serum albumin (BSA). Diluted mouse sera (containing putative antibodies to the amidating enzyme) were then incubated in an amidating enzyme-coated plate and washed. The second antibody (labeled with the tracer, alkaline phosphatase, which facilitates confirmation of binding of the first antibody to the plate bound to the enzyme) was then incubated in the wells. After washing and adding the substrate solution, the signal was monitored colorimetrically with a spectrophotomeric recorder. The signal to noise ratio of "positive" sera was at least 2: 1.

Twenty-seven mice with a higher titer in ELISA were sacrificed four days later. ·. after confirmation of its immunization. The spleen was removed aseptically and obliterated (mechanically crushed) to give a total of 132.6 x 10 splenocytes, which • · [· .1. was combined with 122.8 x 10 6 NS-1 myeloma cells using 1.28 mL of PEG (polyethylene glycol) 4000. The cells were aliquoted into five 24-well plates which were «» »· 1 · 1: 25 coated Balb / cJ thymocytes and splenocytes which function as mast cells. The cells were stored in selective HAT media, which allow only hybrid cell survival.

The supernatants from 116 wells showing clonal growth were analyzed by radi: V: 30 oimmunoassay and ELISA for antibody production. : no analysis was similar to the ELISA above, except that the second antibody was labeled I and radioactive calculations were performed on a gamma counter.

Of the 42,116 wells, 56 were positive for antibody production and subsequently analyzed for reactivity for alpha amidating enzymes. 25 clones which appeared to be positive for alpha amidation enzymes were cloned by serial dilution. Primary clones were analyzed for both alpha amidation enzymes and BSA (since the polystyrene plates were blocked with BSA, the antibodies bound to the plate could only bind to BSA or were absorbed non-specifically) to determine whether they were really specific for the amidating enzyme. Clones possessing a signal for an alpha amidating enzyme at least twice as large as the signal directed to BSA were cloned at a ratio of 1 cell / 2.

In the tertiary cloning step, 21 positive hybridomas (see Table III) were made and 20 of them were characterized by the antibody class by both Ouchterlony and ELISA. Seventeen of the clones had IgG2a heavy chains and three had 15 IgG1 heavy chains. All twenty clones were determined to secrete antibodies with kappa light chains.

Each line is individually grown in bulk culture and cell sections frozen in liquid nitrogen. Pristan pretreated Balb / cJ mice were injected intraperitoneally with 5 x 10 6 hybridoma cells. One week later in mice that did not occur. ascites tumor, boosted by cellular injection. Ascites fluid and blood were removed after 1-2 weeks. After treatment, ascites and sera were analyzed and titrated for alpha-amidating enzyme as well as for negative antigens (e.g.,:: bovine serum albumin, protein, carbonic anhydrase and growth hormone releasing α · 1 · 25) for antibody specificity.

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Table III

43 110433

Balb / c alpha-amidating enzyme monoclonal cell lines 5

Cell Line Heavy Chain Titer1 4-1-4-20-3 IgG2a -1: 5,000 4-1-4-18-1 1gG2a 1 ° 4-5-7-3 19G2a -1: 5,000 4-2-3-17-7 igG2a 3- 7-1-20-24 19G2a -1: 10,000 4- 8-15-2-6 IgG2a 4-7-21-14-9 ig ^ a 4-10-45-1-16 igG2a 15 54- 2-1-39-2 IgG2a -1: 10,000 4-6-12-3-17 19G2a 52-1-31-24-2 IgG2a 4-3-11-46-1 IgG2a 3- 7-9-9- 3 IgG2a 86-1-38-15 IgG, 1: 1,000 20 ^ 4- 5-6-1 IgG ^^ 1: 1,000 4-4-24-3-5 IgG2a -1: 5,000; v 4-H-3 -1-8 IgG1 '8-9-94-4-2 IgG2a: 2 4-10-30-3-1 IgG2a: 75-10-17-14-4 IgG- 25 y 2a' · 92-10-26 -32-39 Not classified * 2 · • · · * · · * 2 »2 •» »· · 30 t» »· ·, ·. : 1 Ascites fluids were titrated with 100 ng of pure enzyme in solid phase ELISA.

44 110433 B. Method for Purifying Monoclonal Antibodies in Mice

The α-amidation enzyme-specific monoclonal antibodies prepared by the above methods are purified as follows. Ascites fluids collected from several mice inoculated with the same clone were used as the antibody source. The ascites fluid was diluted (5-fold) with 10 mM MES, pH 5.6. The diluted ascites fluid was applied to a 1.5 x 20 cm column containing 40 µm ABX mixed silica resin (J. T. Baker) previously equilibrated with 10 mM MES buffer, pH 5.6. Monoclonal antibodies were eluted from the column using a gradient of 0 to 500 mM sodium acetate, pH 7.0. Fractions containing purified antibodies were pooled, tested for specific activity and stored at 4 ° C for later use.

C. Production of Polyclonal Antibodies Specific to α-amidation Enzymes in Channels

Intravenous, intramuscular and subcutaneous injections were given to two laying hens using a total of about 50 µg of purified α-amidating enzyme in Ri-biadjuvant for each chicken. The Ribi adjuvant is a fully metabolizable lipid emulsion system consisting of mitogen and adjuvant chicken lymphocytes to improve antibody response to poultry (Ribi Immunochem t :: Research, Montana). After booster immunization, two booster injections were given. Weekly using about 30 pg enzyme per chicken. The animals were bled: on day 21 and day 35 and sera were processed and analyzed for the presence of specific antibodies, • both chickens sera, 0.

Day 1 (preimmune), day 21 and day 35 were analyzed by solid-phase ELISA against 100 ng of purified α-amidation enzyme. Bovine serum albumin was used as a * * * negative control for nonspecific antibody binding. Antibody specific antibodies were detected with alkaline phosphatase labeled with rabbit chicken IgG. · 30 sera.

t »1 '·': The results of the above methods indicated that specific antibodies could be detected in t 25 serum of chicken 25 days. At a ratio of 1: 10,000 diluted serum 110433 45 produced a signal to noise ratio of about 4: 1. Chicken 258 showed enzyme-specific antibodies on days 21 and 35. Serum diluted 1: 10,000 from both blood draws produced a signal to noise ratio of about 4: 1. Collecting eggs from both of them 5 of them were launched on the 6th. IgYs isolated by polyethylene glycol (PEG) precipitation of preimmune eggs and eggs after immunization were analyzed by Ouchterley methods and enzyme specific antibodies were analyzed by ELISA.

D. Purification of Chicken IgY Antibodies Bacterial polyclonal antibodies specific for the? Α-amidation enzyme were prepared in chickens as described above. Immunized chicken eggs were harvested and either immersed in paraffin or frozen until used to purify IgY. Egg whites were separated from yolks containing antibodies specific for α-AE. Egg yolks were diluted 3-fold using 10 mM sodium phosphate, pH 7.5, containing 0.1 M NaCl and 0.01% sodium azide. The initial PEG precipitation step was performed using a final concentration of 3.5 of PEG 8000. The precipitate was allowed to form for 30 minutes at room temperature, then centrifuged and the supernatant (containing IgY) was saved. More PEG was added to the supernatant to bring the final concentration to 12.5% PEG. IgY antibodies at this concentration of PEG were precipitated by centrifugation and centrifuged. The IgY antibodies of this purification step were further purified by two methods: 1) The precipitated IgY antibodies were resuspended in 10 mM MES, pH 5.6, then • · T ί {dialyzed overnight at 4 ° C against this same buffer. The sample was then loaded on a 1.5 x 20 µM 25 cm column containing 40 µm mixed ABX silica resin (J. T. Baker). The following purification step was the same as that shown for the ascites fluid.

2) Alternatively, the IgY containing pellet was resuspended in the initial buffer and

Ml • I

• * · 'was then reprecipitated using saturated ammonium sulfate (3: 1 v / v).

Pellet containing 30 IgY was resuspended in a small amount of distilled H 2 O and

Ml: ... · was stored at 4 ° C for later use. The chicken IgY immobilization procedure I * * * is shown in Example 12.

»* T i i • I I • ·« 110433

Example 9

Kinetic studies of alpha amidation enzyme activity

The alpha amidating enzyme, purified to homogeneous (both from CA-77 medullary cultures of KIL-5 carcinoma carcinoma and the corresponding tumor tissue removed from the laboratory rat), acts to convert inactive glycine-expanded peptide pro-hormones to bioactive C-terminal amides. Based on our studies, the C-terminal amino acid of the prohormone must be a glycine residue to be recognized by the enzyme and amidated as follows: io amidating enzyme RX-Gly-COOH + O 2 -> RX-CONH 2 + HCO-COOH + H 2 O strictly in addition to the peptide substrate, molecular acid and a reducing agent (L-ascorbic acid). However, we have found that the enzymatic activity can be substantially increased by the addition of Cu + 2 ions (copper sulfate) and catalase. This exogenous copper is bound by an enzyme and used as a site for binding and activation of molecular oxygen. On the other hand, catalase is an ent-20 enzyme that acts to flush hydrogen peroxide that could otherwise accumulate on the sidewall. * · :: due to actions requiring oxygen, ascorbate, copper, which could destroy «·! * · *: Amidating enzyme.

We have successfully developed sensitive non-radiometric assays to detect alpha- • · ·: 25 amidating enzyme activity. Synthetic tripeptide substrates are used in these assays. The amidation of these substrates is preferably controlled by separators. *: bat and qualitatively determine the product (amidated dipeptide and substrate tripeptide) • · · * using HPLC. The most sensitive of the assays developed is • · v .: monodansyl-L-Tyr-Val-Gly. This compound can be detected at very low levels because the dansyl group is fluorescent. Thus, the sensitivity of this assay can be compared to similar radiometric assays developed in other laboratories.

• · «· · * · · 110433 47

We have used this assay to study the kinetic properties of the alpha amidating enzyme. In particular, the kinetic parameters Km and Vmax have been determined by studying the effect of varying the substrate concentration on enzyme activity. Km is a measure of the affinity that an enzyme has for a particular substrate. The lower the Km, the lower the affinity of the pie-5. Vmax is the maximum rate at which the enzyme converts sub-substrate to product and is detected at substrate saturation concentrations (i.e., sub-substrate is present in a high excess relative to the enzyme).

| j i

In a typical amidation enzyme assay, the reaction system (50 µl) would comprise enzymes (about 7 µg), dansyl-L-TyrVal-Gly (up to 40 µΜ), L-ascorbic acid (3 mM), catalase (0-100 µg ml). 1) and copper sulfate (0-2 µM) in 60 mM TES buffer, pH 7.0. The reaction is initiated at 37 ° C by addition of enzyme and terminated after a specified time by addition of 0.1 M EDTA (final concentration) which binds copper by rendering it unavailable for enzyme.

15

The alpha amidating enzyme has a high affinity for the enzymatic amidation of dansyl-LTyr-Val-Gly with a Km of 1-2 pM. This value seems to be constant despite the degree of purity of the enzyme. Thus, the enzyme stock that is derived

Sephacryl S-300 from chromatography, has the same Km as the enzyme electrophoretically; ; 20 pure preparations derived from Mono Q chromatography (pH 8.0) or lower; · · · · / ··; of molecular weight analyzed by Mono Q chromatography, pH 6.0. On the other hand, as expected, Vmax values (expressed as mg of protein):: *: vary substantially with the degree of purification. Thus, when the Vmax of the depot derived from Sephacryl S-300 is about 50-100 nmoles of product / min / mg protein, the Vmax of pure morphine enzyme is about 5000 nmoles of product / mg protein.

• · • tl •. ·: We have found that ascorbate and substrate can compete with each other for enzyme. · 'In some circumstances. For example, if the substrate concentration is substantially increased above the saturation level, the enzyme activity is diluted due to a weakened interaction between the enzyme and ascorbic acid. Thus, for each specific substrate, there appears to be a fine balance between optimal substrate / ascorbate levels, depending on the affinity of the enzyme for the particular substrate.

f 48 110433

Affinity of amidation enzyme to glycine-extended peptides

We have used monodansyl-L-Tyr-Val-Gly amidation assay as a sensitivity probe for the reaction between the amidation enzyme and several Gly-extended peptide prodrugs. The purpose of this study was to investigate the relative affinity of an enzyme for binding to certain types of peptide substrates. Although we have previously shown that the enzyme successfully amidates precursor substrates expanded with human calcitonin and human growth hormone releasing factor glycine, this does not indicate any ability of the enzyme to bind these or other peptides to one another.

We have shown that glycine-expanded alpha-melanocyte stimulating hormone, substrate P, vasopressin analogues, and growth hormone releasing factor interact with high affinity for the amidating enzyme to prevent it from metabolizing dansyl-LTyr-Val-Gly. Further, the pentapeptide models corresponding to the five C-terminal amino acid residues of human glycine-expanded calcitonin, neuropeptide Y, cholecystokinin, corticotrophin-releasing factor, and calcitonin gene-related peptide show competition in this assay. In other words, all of these glycine-expanded peptide substrates are capable of binding and presumably amidating the enzyme.

t I · * • · • · · f ·

In contrast, when investigated amidated peptides corresponding to these substrates, they are found to be much less capable of interacting with the enzyme, i *: The capacity of the catalytic site of this enzyme to recognize a large number of glycine-la: 25 substrates renders it very useful. commercial amidation of recombinant peptide prodrug.

«·

EXAMPLE 10 Isolation of α-amidation enzyme peak III coding for DNA sequence coding for DNA sequence 30: * · ': Total RNA was prepared from rat MTC tissue using the guanidine thiocyanate method. Poly-A RNA was selected on oligodT cellulose.

49 110433 cDNAs

The double-stranded cDNA was prepared by well known methods. Using rat MTC-derived poly-A-RNA as a template and oligo-dTi2-i8 as a template, the first fiber synthesis was performed in an enzymatic reaction by reverse transcription. cDNA and RNA were separated and RNA was digested with alkali. The second strand synthesis of cDNA was self-initiated using E. coli DNA polymerase I. S1 nuclease digestion was used to remove hairpin loops from the cDNA and to cleave the single stranded regions of the cDNA. Following reaction with DNA polymerase I, to generate flat ends, the double-stranded cDNA was treated with EcoRl methylase and s-adenosylmethionine to methylate the EcoR1 sites and protect them from subsequent enzymatic cleavage. EcoR1 binders were fused to the cDNA. After EcoR1 digestion, the excess binders were separated from the cDNA and the cDNA was fractionated on a Sepharose 4B column. For one such synthesis, molecules larger than 500 base pairs were collected, while in another, molecules larger than 1000 base pairs were pooled for cloning.

λ gtl 1 Construction of a cDNA library. ·. Following synthesis of the double-stranded cDNA bound by the linkers, the molecules were used

':.; To produce cDNA kiqastases in vector Xgt 11. This was done by inserting the cDNA

; * ·; Xgt11 DNA cleaved with Eco RI and treated with phosphatase to prevent vector DNA: *: self-assembly. After DNA insertion, the recombined DNA was packaged in vitro to produce infectious bacteriophage particles. (Packaging extracts::: 25 are commercially available from Promega Biotech or Clontech Laboratories or may be prepared by standard procedures).

• · * • · ... * After compression of DN A, portions of the compression mixture were tested to determine the amount of recombinant Y: banners. One library was found to contain about 2.57x106:,: 30 infectious particles, of which about 78% were obvious recombinants (forming clear '' '; plaques on X-Gal plates grown in the presence of IPTG). Another library found: * ·. I containing about 2.75 x 106 total plaque forming units and about 81% apparent recombinants.

110433 50

Library analysis

To identify which recombinant bacteriophage contained the cDNA of the alpha amidating enzyme 5 protein, the phages were analyzed with radiolabeled oligonucleotide probes formed from the specific amino acid sequences of the alpha amidating enzyme. (See Example 7, Table II). Analysis was performed by coating bacteriophage samples and lifting the phages onto cellulose nitrate filtration discs. Methods for immobilizing phages on cellulose nitrate filters are widely known. Dual filters from a 10-well plate were hybridized with 32P-labeled oligonucleotide AE 9. Hybridization was performed at 37 ° C for 20-24 hours, 6 x NET, 0.5% NP40, 5 x Denhardt's solution, 100 pg / ml salmon sperm DNA with an oligonucleotide probe at 0.3 to 0.4 pmol / mg. After hybridization, the filters were washed at 6 x SCC 44 at 45 ° C for several hours and X-ray film. Positively hybridizing phages were identified as uniform spots on the double filters. These were purified by enrichment in successive rounds of coating and hybridization. Of the 4-5 x 10 4 phages analyzed, 18 were identified by AE 9.

To compare specificity in the selection, hybridization was performed with another alpha-20 amidation enzyme nucleotide, AE 8. This test showed that at least four • »11 · •.:. of the dexed phage contained the cDNA of the alpha-amidating enzyme protein sequences. This was confirmed by additional oligonucleotide hybridization (AE 4 and AE 5) as well as by DNA sequence analysis.

The expression of the 25 α-amidation enzymes, the peak IIIa AE for which we have determined the protein sequences, has a molecular weight of about 75,000 daltons. If the average molecular weight of the amino acid is 120: V: Dalton, then the amidation enzyme will have a maximum of 625 amino acids. The 625 amino acid: 30p gene must contain at least 1875 base pairs. All four cDNAs: v. Which we have isolated as specific for the amidation enzyme, are large enough to encode: "·. · Completely α - amidation enzyme protein. One of the cDNA clones, λ AE 1, is n.

2,200 nucleotides. At the first 50 nucleotides at its other end, it begins encoding 110433 51 for the amino acid sequences identified as the amino terminus of the peak III enzyme. Therefore, it can be concluded that the cDNA contains all the coding capacity required for the peak III enzyme. Known for this information and nucleotide sequences at the ends of the 2,200 bp cDNA, it is relatively simple to insert 5 cDNA for genetic cloning into a prokaryotic host such as E. coli.

One possible method that can be applied to λ ΑΕΙ cDNA is shown in Figure XXXII. For example, a 2,200 base pair cDNA adapter of λ ΑΕΙ is isolated after Eco RI digestion and agarose gel electrophoresis of recombinant bacteriophage DNA.

It is cloned into the EcoRl site of pBR322 to produce plasmidAE8-1. pAE8-1 contains a unique KpIll cleavage site in cDNA sequences and a unique HindIII site in pBR322 sequences. Digestion of pAE8-1 with KpnI and Hind III produces n.

A 2.15 kb fragment which has lost about 62 bp of the cDNA (coding end of the amino terminus of the cDNA). To recover the amino acids found at the amino terminus of the hui-15 red III amidation enzyme and to adapt the cDNA for cloning into the expression plasmid, the Kpn1-Hind III-terminated fragment is ligated to oligonucleotide binding adapters. The example shown uses the E. coli detection plasmid pHH233-2 provided by Pharmacia. The 2.15 kb cDNA fragment is ligated into a double-stranded binding adapter consisting of. 20 ..:: 5, 3 '. ·· * ·: AE17 (+) 3 0 CATGTCATTTTCCAATGAATGCCTTGGTAC: ta:' v and '·' · E '3' ::: AE18 (-} 2 2 CAAGGCATTCATTGGAAAATGA: at a .

• · · • · • · V: 25

The inserted fragment is then inserted into the plasmid pKK233-2 with the DNA cleaved above. '·: With NcoL and Hind III to produce a 4.6 kb linear vector. The coupled product, pAE12, contains the alpha-amidation enzyme cDNA preceded by the ATG start codon • ·, :: and the ribosome binding site and, under hybrid control, the IPTG-inducible promoter, trc. Gene- 30 are followed by the 5S gene and the transcription termination site. The IPTG-inducible il-'* *: taste of pAE12 is obtained after transformation of plasmid DNA into E. coli strain by the laciq genotype.

52 110433 λΑΕ1: η Partial DNA Sequence of the 2.2 kb cDNA Adapter The 2.2 kb insert is removed by EcoR1 thawing of λ AE1 and the insert is designated 32p. The 1.6 kb and 0.6 kb fragments obtained after secondary digestion with Hinc 11 are sequenced by the Maxam and Gilbert chemical degradation method.

DNA sequence obtained by Maxam-Gilbert sequencing of the 600 kb fragment of the λ AE-1 EcoRl end: 10 v v v v 50v aattccggtctttaagaggtttaaagaaactaccagatcattttccaatg v v v vOOv

AATGCCTTGGTACCATTGGACCAGTCACCCCTCTTGATGCATCAGATTTT

v v v v 150v

GCGCTGGATATTCGCATGCGTGGGGTTACACCTAAAGAGTCTGACACATA

v v v v 200v

CTTTCTGCACGTCCATGCGTCTACCT

20 DNA sequence obtained from the 1600 kb fragment of EcoR1-end λ AE-1 by Maxam. '· * ·: Gilbert Sequencing: i «» vvvv 50v

25 AATTCCGTCTCAGTTTCTGTTTCTCTTGCATCTTCTGCAATTCTGAGGAG

v v v v lOOv

: '·. · GTGGGTTTGTTCTCCACTTTGGGTTCGACAACTGCCTCGGCTTCTTTG AT

'... · v v v v 150v

TTCGTGGACTTCGATGCCAGCCTTTTTAACTGACGCATGCTCCATTTTTT

• # ·, ···. v v v v 200v

30 CGGTCAGGGTGAACTTCCACACGGTGTTGTGTGTGCGCTCGAAGACCG

110433 53 i j

Investigation of sequence homology with oleaonucleotide probe AE8 (-) 22

A computer-based study was performed to examine the homology between the translation of the amino acids used to produce the AE8 (Leu-Gly-Thr-IleGly-Pro-Val-Thr) probe and the λΑΕ1: η 600 kb fragment of DNA.

A region of complete homology was obtained and this region is indicated by asterisks in the DNA sequence and capital letters for amino acids. The amino acids identified by NH2-terminal sequencing of the purified amidation enzyme are shown in brackets. Amino acid presentations found to differ from those predicted by the DNA sequence are indicated by

AATTCCGGTCTTTAAGAGGTTTAAAGAAACTACCAGATCATTTTCCAATG

---- · ---- + ---- + ---- · ---- · ---- · ---- + ---- + ---- 1 ---- + so ipvfkrfkettr (sfsne 15 + **********************

AATGCCTTGGTACCATTGGACCAGTCACCCCTCTTGATGCATCAGATTTT

---- · ---- + ——— · ---- + ---- 1 ---- + ---- 1 ---- + ---- · ---- + JOO

C LGT IGPVTpl dasdf ++

GCGCTGGATATTCGCATGCCTGGGGTTACACCTAAAGAGTCTGACACATA

—--- 1 ---- + ---- 1 ---- + ---- 1 ---- + ---- 1 —— + ---- 1 ---- + 150 20 aldirm.p) gvtpke sdty * '· · +

CTTTCTGCACGTCCATGCGTCTACCT

---- · ---- + ---- 1 ---- + ---- · - 176 .1 flhvhast.

• · 1 t t 1 • · · · · · * 1 · • · •:: · 1 25 Example 11 • "-

Immobilization of the Enzyme • 1 1 • <· • · t · »

• I

.. · 'a-AE purification: 1 »• · · • ·:.,. Prior to immobilization, the α-amidation enzyme was purified by weak anion exchange and gel V '': additional filtration chromatography (Example 4). In some cases, the enzyme preparation t # '· · can be further purified using either immunoaffinity chromatography or phenylsepharose chromatography. The subsequent immobilization method is 110433 54 independent of the purification method, but usually the specific activity should be at least 25 mU, and in particular 50 mU or higher.

Immobilization of α-AE: The 5-α-amidation enzyme immobilization technique is based on the simultaneous reaction of three components, an enzyme, a water-soluble copolymer of acrylamide (PAN) and a low molecular weight crosslinking reagent (TET). The prefabricated polymer (PAN) consists of acrylamide and N-acrylosuccinimide polymerized in THF-10 solution using a thermal start with azobic (isobutyronitrile). The crosslinking reagent may be α, ω-diamine, triethylenetetramine, (TET) cystamine. Reaction of the diamine with PAN active ester groups crosslinks the polymer chains through amide bonds and forms an insoluble gel. The amino functions of the enzyme (preferably the ε-amino group of lysine) react simultaneously with the remaining active esters in gel 15 to form stable covalent amide bonds. Immobilization is carried out in the presence of substrate and cofactors. The presence of a high affinity substrate and co-factors at concentrations higher than Km inhibits reactions between PAN active esters and nucleophilic groups at or near the catalytic site of the enzyme, thus protecting the enzyme from chemical inactivation.

20 hrs · · ·

Immobilization conditions • · · · t | The partially purified α-AE is made soluble in 30 mM HEPES buffer, pH 7.0.

• · I

• The ratio of PAN to TET is formed such that 15% of the active esters remain unreacted and thus act as α-amidation enzyme binding sites. The standard PAN solution is 20% (w / w). The α-amidation enzyme reaction mixture consists of 0.2 μΜ CuSO 4, 40 μΜ dansyl-His-Phe-GIy and 10 mM ascorbate 0.5-2.0 mg α-ΑΕ / g PAN.

The concentration of TET is calculated to be equal to 0.85 equivalents of primary * * · '' primary amine / equivalent active ester. Usually, to determine the optimum conditions, the reaction without enzyme is carried out for a gel time. optimization, i.e., the time required after addition of TET to obtain gel formation, optimization yields of amidation enzyme immobilization are obtained when the addition of the enzyme is moved closer to the gelation point. As a general rule, the shorter the duration of enzyme exposure to PAN prior to gel formation, the higher the yield of active immobilized enzyme. For most reactions, α-AE (about 2.0 mg / g PAN) is added 45 to 60 seconds after TET. The enzyme-containing gel is allowed to stand at room temperature for about 1 hour to allow completion of the coupling reaction. After 60 minutes, the gel is ground with a mortar and pestle to give fragments of average size 100 μ. These particles are washed with ammonium sulfate to remove unbound starting materials and to convert the remaining active ester groups to amides, thereby covering the reactive groups. The yield of the active α-amidation enzyme after immobilization is expected to be less than 60%. Post-immobilization washings may contain 30-40% of initial activity. The α-amidating enzyme can be recovered from the wash waters by increasing the concentration of ammonium sulfate to 45%, which causes the active α-amidating enzyme to precipitate.

Immobilized gel particles are suitable for a periodic amidation reaction in which the particles are suspended in a mechanical stirrer. When the enzymatic reaction is complete, the particles are allowed to settle and the supernatant containing the amidated peptide product is decanted. This method is not optimal for large scale reactions. Enzyme-containing gel particles are not rigid enough to be packaged 20 sieve and provide reasonable flow rates. There are two alternative ways to eliminate this problem: 1. The gel is allowed to polymerize in the presence of glass beads. Usually minutes before • · · * * ·. The 'gelation point' glass beads are added to the reaction mixture and mixed with a mechanical stirrer until the beads are covered with a layer of PAN solution. The flow properties of this mixed material are much better than those of the gel particles alone. * Background Features.

Alternatively, the PAN particles are mixed with a filter aid, Celite 545.

> · · # * '·; Usually, a mixture of PAN and Celite is prepared with a PAN of less than 8% w / w (dry weight). To form a column of this type, a gel particle. '*: The cartridges are suspended in 50 mM Tris: HCl, pH 7.0, and Celite 545 is added with continuous stirring and stirred for an additional 2 hours. The column is packed with this slurry (3 x 40 cm) and the temperature of the 110433 56 column is maintained at 37 ° C to facilitate the amidation reaction. Using this method, the flow rate is maintained at 8-101 / day.

An interesting alternative to column chromatography is to use a tangential flow system. The PAN polymer is poured onto a 0.45 µm poly-sulfone pore support plate before the gelation point. After gelling, the plates can be cut to fit Millipore or New Brunswick ultrafiltration units of tangential flow. In this model, polysulfone PAN immobilized enzyme gel mixed sheets are stacked in layers to provide a huge increase in surface area. This method can be directly increased to flow rates of liters per minute. This type of system also provides a convenient method of circulating the reaction mixture to maximize amidation of the peptide substrate.

Advantages achieved by immobilizing the α-amidation enzyme include the recovery and reuse of the enzyme. Second, the immobilized matrix increases the stability of the enzyme and provides an enzyme treatment form that can be used in large-scale amidation reactions (ranging from grams to kilograms of substrate) over extended time periods.

Example 12 Preparation of Immunoaffinity Peptide for Purification of Impure Alpha-Γ **: Amidation Enzyme Compositions Compositions A. Immobilization: • · "'" it "*" · The immobilization matrix used was cyanobromide-activated Sepharose 4B (Pharmacia).

The dry gel was first washed with 1 mM HCl (200 mg / g resin) to expand the solid support and wash. About 40 mg purified polyclonal antibody purified

• I

V, · chicken egg yolks were dialyzed against 100 mM HaHCO 3, pH 8.3 containing

Ml 1: 30 0.5 M NaCl. Coupling was performed using 8 ml of the above expanded and washed solid.

; ' * 'support. The reaction was carried out at room temperature for 100 hours at 100 mM

: * *. in sodium bicarbonate buffer, pH 8.3, containing 500 mM NaCl, which was used to prevent non-specific binding of the protein to the solid support. The remaining 110433 57 active groups of the gel were blocked using 0.2 M glycine. After the blocking step, the gel was washed 4 to 5 times using a cycle of high and low pH buffers (high pH buffer 100 mM NaHCCb pH 8.3 + 500 mM NaCl, low pH buffer 100 mM acetate pH 4.0 + 500 mM NaCl). These 5 washing steps removed the unbound protein and blocking reagent (glycine) from the resin. The immunoaffinity resin was stored at 4 ° C in a basic pH buffer containing mertiolate as a bacteriostatic agent. All subsequent immunoaffinity chromatography is performed at 4 ° C. A similar method can be used to immobilize purified monoclonal antibodies.

10 B. Immunoaffinity chromatography

The immunoaffinity column was used as an alternative highly efficient step in purifying α-AE. Tissue culture medium from CA-77 cells (see Example 15 4) is diluted with distilled water and pumped through a DEAE anion exchange cartridge previously equilibrated with 20 mM Bis Tris-HCl pH 6.0. The α-amidation enzyme is eluted from the cartridge with 50 mM Tris: HCl pH 7.0 containing 500 mM NaCl. Fractions containing α-AE activity are either dialyzed against Tris-HCl buffer, pH 7.0, or further purified using gel filtration chromatography (Example 4) before. ·. 20 immunoaffinity chromatography. Samples containing a-AE are controlled by immunoadsorp- •.:. through a thi-column at neutral pH. Antibodies specifically bind the α-amidation enzyme, while impure proteins do not bind and are removed in eluent. a-:: 1: AE activity can be eluted from the column using 100 mM glycine: HCl buffer • · · pH 3.0 (other desorbents may be used, including urea, dioxane, ethylene glycol and Nalt). ). Fractions are collected in 1.0 M Tris: HCl pH 7.0 to purify the buffer system while maintaining α-AE activity.

• · · • • • 1 • 1 • 1 • · · • t # · • · 58 110433

Summary of Possibilities of Utilizing the Invention 1. An enzymatic composition comprising at least one alpha-amidating enzyme capable of catalyzing the conversion of a peptidyl compound having a glycine residue to a C the enzymatic composition is sufficiently pure in alpha-amidating enzymes to have a specific activity of at least about 25 mU / mg protein, and the enzymatic composition is sufficiently free from proteolytic activity capable of cleaving at least one entity, the peptidyl compound, and alpha-amidating enzymes such that said composition can catalyze the conversion when the peptidyl compound contains L-amino acids.

Preferably, the enzymatic composition contains at least one alpha-amidating enzyme that may be obtained from medullary thyroid carcinoma tumors, for example, rat medullary thyroid carcinoma tumors.

Preferably, the enzymatic composition contains at least one alpha-amidating enzyme which can be obtained from cell culture medium derived from IVI-10029 (later ATCC CRL 10919).

»·» ♦. · ·: 2. An enzymatic composition comprising at least one alpha amidating enzyme which. ': is able to catalyze the glycyl • · • in the presence of oxygen and a reducing agent at least at its C-terminus. · · Conversion of a peptidyl substrate containing a residue to the corresponding peptidyl amide having • ': 25 amino groups at the site of said C-terminal glycine with an alpha amidating enzyme

• M

:: an apparent molecular weight of about 60,000 to about 65,000 daltons as measured by gel filtration chromatography and about 73,000 to about 75,000 daltons as measured by electrophoresis on sodium dodecyl sulfate / polyacrylamide gel that the alpha-amidating enzyme · omega · elute; sodium chloride solution from an anion exchange column (equilibrated at pH 6, particle size, · · 30 cascade size 10 µm, cavity volume 40%, and gel with charge group -CHh-hT- (0¾¾ and: ... ·) with ionic capacity of 0.28-0.36 mmol / ml) with pH 6 eluting first at a concentration of sodium chloride of about 220 mM or greater than that of alpha amidating enzyme · ·: eluting with a pH 8 sodium chloride solution anion exchange column (equilibrated at pH 8), wherein the pH 8 elution is first performed at a sodium chloride concentration of 11043359 at or above 250 mM, and that the optimal activity of the alpha amidating enzyme is at pH 4.5-5.5 (as measured by the fraction of dansyl-L-Try-Val-Gly converted to dansyl-L-Try-Val-CONH2 in 16 minutes) that the alpha amidating enzyme is sufficiently pure in the alpha amidating enzymes , so that the specific activity of the enzymatic composition is at least about 25 mU / mg protein.

The alpha-amidating enzyme of the above-mentioned enzymatic composition has an isoelectric point of about pH 4.8 as determined by a non-denatured isoelectric focusing acrylamide gel, and the N-terminal amino acid sequence of the alpha-amidating enzyme is 10 12 3 4 NH 2 -Ser-Phe-Ser-Asn.

3. An enzymatic composition comprising at least one alpha-amidating enzyme or an active fragment or homologue thereof comprising a peptide chain substantially homologous to at least one of the following amino acid sequences:! 20! . ·. (a) *; V -Ser-Phe-Ser-Asn-Glu-Cys-Leu-Gly-Thr-Ile-Gly-Pro-

Val-Thr-Pro-Leu-Asp-Ala-Ser-Asp-Phe-Ala-Leu-Asp-Ile- '· 1 Arg-Met-Pro • 1 • · · :: v (b): 1! 25 -Ser-Met-Gln-Pro-Gly-Ser-Asp-Gln-Asn-His-Phe-Ser-'Gln-Pro-Thr-Vi (c) • · · -Asn-Gly-Gln-Trp-Thr -Leu-Ile-Gly-Arg- 30 '! 1 -Phe-Val-Thr-Gln-Trp-Gly-Glu-, t 1 »

• I

110433 60 and the composition is sufficiently pure in the alpha amidation enzyme so that the composition has a specific activity of at least about 25 mU / mg protein and is sufficiently free from proteolytic activity capable of breaking down at least one whole such as the substrate peptidyl compound, the corresponding peptidyl amide product and the alpha amidation enzymes so that the composition can catalyze the alpha amidation reaction when the peptidyl compound contains L-amino acids.

An enzymatic composition comprising at least one alpha-amidating enzyme capable of catalyzing the conversion of a peptidyl substrate containing a glycine-10-containing peptide to a corresponding peptidyl amide having an amino group at the C-terminal glycine amine at least at its C-terminus. having a molecular weight of about 60,000 to about 65,000 daltons as measured by gel filtration chromatography and less than about 55,000 daltons as measured by electrophoresis on sodium dodecylsulfate / polyacrylamide gel, eluting the alpha-amidating enzyme with pH 6 10 µm, 40% cavity volume and gel with a charged group of -CH 2 -N * - (CH 3) 3 with an ionic capacity of 0.28 to 0.36 mmol / ml), eluting first with a concentration of sodium chloride of n 160 mM or more, and that alpha-amido the optimal activity of the enzyme is at pH 7.0-8.5 (as measured by the 20 fractions of dansyl-L-Tiy-Val-Gly converted to dansyl-L-Try-Val-CONH2 in 16 minutes) that the enzyme -: *: the matrix composition is sufficiently pure in alpha-amidating enzymes so that the enzymes-

f · · I

. The specific activity of the '·' composition is at least about 25 mU / mg protein.

t »· • · • • •. 5. An enzymatic composition comprising at least one alpha amidating enzyme which: *. · 25 is able to catalyze the presence of oxygen and a reducing agent at least at its C-terminus glycyl * · ·:. * * conversion of a peptidyl substrate containing a residue to the corresponding peptidyl amide having an amino group at the site of said C-terminal glycine and having an alpha amidating enzyme. *: apparent molecular weight of about 60,000 to about 65,000 daltons as measured by gel filtration chromatography and less than about 55,000 daltons as measured by sodium dodecyl electrophoresis. ·: 30 on a sulfate / polyacrylamide gel eluting the alpha amidation enzyme with a pH 6: ... · sodium chloride solution from the anion exchange column (equilibrated at pH 6, • '·': particle size 10 µm, 40% cavity volume and gel charged group is -CH2-N + - (CH3) 3: with an ionic capacity of 0.28-0.36 mmol / ml), with pH 6 elution first at a concentration of ca. 160 mM sodium chloride or its and that the alpha amidation enzyme is 61 110433 (eluted with a pH 8 sodium chloride solution from an anion exchange column (equilibrated at pH 8), with pH 8 elution first at a sodium chloride concentration of about 185 mM or more). , and that the optimum activity activity of the alpha-amidating enzyme is at pH 5.0-8.5 (as measured by the fraction of dansyl-L-Try-Val-Gly which is a modified dansyl-L-Try-Val- CONHf in 16 minutes) that the alpha amidating enzyme is pure enough such that the specific activity of the enzymatic composition is at least about 25 mU / mg protein.

An enzymatic composition comprising at least one alpha-amidating enzyme capable of catalyzing the conversion of a peptidyl substrate containing a glycine residue to a corresponding peptidyl amide having an amino group at the position of said C-terminal glycine and at least at its C-terminus. having a molecular weight of about 60,000 to about 65,000 daltons as measured by gel filtration chromatography and about 73,000 to about 75,000 daltons as measured by electrophoresis on sodium dodecyl-15 sulfate / polyacrylamide gel, the alpha-amidating enzyme is eluted at pH 6 with 6 , 10 µm particle size, 40% cavity volume and gel with a charged group of -CH 1 -BT- (CH 3) 3 with an ionic capacity of 0.28-0.36 mmol / ml), with pH 6 elution first [at sodium chloride concentration, 200 mM or more, and that alpha-am elution enzyme is eluted with a pH 8 sodium chloride solution from an anion exchange column (equilibrium: weighted at pH 8) with pH 8 elution first at a sodium chloride concentration of about 220 mM and that the optimal activity of the alpha amidation enzyme is at pH 4.5-5.5 (as measured by the fraction of dansyl-L-Try-Val-Gly which is · ·: modified dansyl-L-Try-Val- CONH2 in 16 minutes) that the enzymatic composition: 25 is sufficiently pure in the alpha-amidating enzymes so that the enzymatic composition • · · '. The specific activity of '1 is at least about 25 mU / mg protein.

• »1 • 1 ': In the above-mentioned enzymatic composition, at least one of al- *; · 1 fa-amidating enzyme has an isoelectric point of about pH 5.8 and alpha-amidating enzyme N-:. The 30 terminal amino acid sequence is 110433 62 NH2-Phe-Lys-Glu-Thr-Thr-Arg or a homologue thereof.

7. An enzymatic composition comprising at least one alpha-amidating enzyme capable of catalyzing the conversion of a peptidyl substrate containing a glycine residue to at least one C-terminal glycine having an amino group at the C-terminus of said C-terminal glycine and apparent molecular weight is from about 60,000 to about 65,000 daltons as measured by gel filtration chromatography and from about 73,000 to about 75,000 daltons when electrophoresed with sodium dodecyl sulfate / polyacrylamide gel, the pH of the alpha , 10 µm particle size, 40% cavity volume and gel with a charged group of -CH 2 -N * - (CH 3) 3 15 and an ionic capacity of 0.28-0.36 mmol / mL) with pH 6 elution first at sodium chloride concentration 240 mM or more that alpha-amidates the enzyme is eluted with a pH 8 sodium chloride solution from the anion exchange column (equilibrated at pH 8), with pH 8 elution only at a concentration of about 250 mM sodium chloride or sufficient enzyme composition to be sufficiently pure in the alpha amidating enzymes. a specific ac-; *: density is at least about 25 mU / mg protein.

• ·

The above enzymatic compositions are sufficiently pure in alpha-amidating enzymes to preferably have a specific activity of at least about 50 mU / mg * * · • *,: 25 proteins, or at least about 150 mU / mg protein, or in some cases at least n.

M »· 1500 mU / mg protein.

• · «*. *: The above enzymatic compositions are sufficiently free from proteolytic activity · · s · »·. * a ability to degrade at least one entity consisting of a peptidyl compound, a corresponding peptidyl amide, and an alpha amidation enzyme so that the composition can catalyze the alpha amidation reaction when the peptidyl compound contains L-amino acids .

| I

• ·: * ·, | A homogeneous purified enzyme preparation containing peptidyl glycine alpha amidating monooxygenase and capable of catalyzing at least the C-terminus 110433 of the peptide chain to convert the peptidyl compound containing 63 glycine residues to the corresponding peptidyl amide having the amino terminus of said C terminus. or prepared by recombinant DNA technology and having a specific enzyme activity of at least 25 mU / mg protein, and the purified enzyme product is sufficiently free from proteolytic activity capable of cleaving at least one entity, the peptidyl compound, the corresponding peptidyl amide and the alpha amidating enzymes, said composition can catalyze the conversion when the peptidyl compound contains L-amino acids.

The homogeneous purified enzyme preparation may have a specific enzyme activity of more than 1500 mU / mg protein.

A purified enzyme preparation derived from medullary thyroid carcinoma tissues, their cell lines or tissue culture media derived from such cell lines, which contains peptidyl-glycine-alpha-amidating monooxygenase and is capable of catalyzing at least the peptide-terminated to the corresponding peptidyl amide having an amino group at the site of said C-terminal glycine, wherein the peptidyl compound or polypeptide is purified from natural sources or prepared by recombinant DNA technology to have a specific enzymatic activity of at least 1500 mU / mg protein and is sufficient: * : free from proteolytic activity capable of degrading the peptidyl compound, · :: peptidyl amide or alpha amidating enzymes so that the composition can catalyze the I '·' conversion when the peptidyl compound contains L-amino acids.

* · · ·: 25 Medullary thyroid carcinoma tissues are preferably derived from rat tissues and have a specific enzymatic activity of at least about 150 mU / mg protein.

·. * · ': Medullary thyroid carcinoma can be, for example, IVI 10028 (later ATCC

* * * 75168) and as cell line IVI 10029 (later ATCC CRL 10919).

: Y: 30 • · ·: 10. Method for purifying at least one alpha amidation enzyme capable of: *. '. to catalyze the conversion of a peptidyl compound containing a glycine residue, · ', ·, at least at its C-terminus, to a corresponding peptidylamide having an amino group in place of said C-terminal glycine, wherein the method comprises protein types having an apparent molecular weight of about 58,000 to about 67,000 daltons, for use in strong anion exchange chromatography using an anion exchange resin capable of binding the alpha-amidating enzyme, and eluting with the alpha-amidating enzyme salt solution.

Strong anion exchange chromatography is preferably carried out after size exclusion chromatography and is carried out on an eluent portion of size exclusion chromatography having the characteristic activity of an alpha amidating enzyme.

! The method may further comprise an additional anion exchange chromatography step before the size exclusion chromatography step and a second anion exchange chromatography step after the strong anion exchange chromatography step.

Either the strong anion exchange chromatography step or the second anion exchange chromatography step can be performed at basic pH and the other at acidic pH.

In the method, the alpha amidating enzyme can be derived by anion exchange chromatography from the culture medium used to grow the medullary thyroid carcinoma cell line. In the method, the medullary thyroid carcinoma tissue is homogenized in aqueous medium, the homogenate is centrifuged, and the supernatant of the spinning is subjected to ammonium sulfate precipitation.

A process for the efficient preparation of an alpha-amidating enzyme composition, wherein: · 25 methods collect at least one crude alpha-amidating enzyme from natural or! v · from the recombined source, subjected to crude enzyme as well as size exclusion chromatography, which is able to distinguish protein types of apparent molecular weight »*» *. from about 58,000 to about 67,000 daltons, to anion exchange chromatography using an anion exchange resin capable of binding an enzyme to elute the alpha amidation enzyme from an anion exchange resin at a concentration of more than 250 mM, provided that the eluted II t :: enzyme is sufficiently pure so that the specific activity of the composition is at least about 25 mU / mg protein.

I I I

110433 65

Preferably said at least one alpha amidating enzyme is selected by selecting a cell line capable of expressing at least one alpha amidating enzyme by collecting the cell culture medium used for cell line growth.

A process for preparing an alpha-amidated peptidyl product comprising reacting an enzymatically effective amount of the peptidyl compound with an enzymatically effective amount of a peptide terminal glycine 10 site. Preferably, the enzyme is derived from an animal tissue extract using medullary thyroid carcinoma tissue, medullary thyroid carcinoma cell lines, tissue culture medium derived from these cell lines, or a combination thereof. The reaction is preferably carried out in the presence of copper ions, or in the presence of oxygen and a reducing agent.

As reducing agent can be used ascorbic acid, ascorbic acid salts, dihydroxy furoate, metallic cyanide compounds and tetrahydropterin, preferably ascorbate.

The reaction is preferably carried out in the presence of at least one compound which is catalase, ethanol, potassium iodide or a mixture thereof, more preferably in the presence of ascorbate, catalase and cupric ions.

For example, the peptidyl compound may be a peptide containing a calcitonin sequence containing a carboxyl-terminal glycine residue.

13. A process for the preparation of calcitonin, which process comprises providing a substrate comprising an amino acid sequence corresponding to at least one type of calcitonin. amino acid sequence, that the substrate sequence has a glycine residue in its? • · *. '· I at the boxyl terminus, and then reacting the substrate in the presence of an alpha amidating enzyme complex ... ... · containing at least one alpha amidating enzyme capable of: *:': 30 in the presence of oxygen and a reducing agent at least at its C terminus: conversion of the peptidyl compound containing the compound to the corresponding peptidyl amide having amino II. *. a group at the site of said C-terminal glycine that the alpha amidating enzyme is sufficiently pure in the alpha amidating enzymes so that the specific activity of the enzymatic composition is at least about 25 mU / mg protein, preferably at least about 50 mU / mg protein. mU / mg protein 66 110433 and is sufficiently free from proteolytic activity capable of cleaving at least one entity, which is a peptidyl compound, a corresponding peptidyl amide, and alpha amidating enzymes, so that the composition can catalyze the conversion when the peptidyl compound contains L-amino acids. More preferably, the enzymatic composition has an activity-5 activity of at least about 150 mU / mg protein.

The substrate can be obtained by a recombinant technique which isolates or prepares a DNA sequence capable of expressing the calcitonin amino acid sequence, attaches the DNA sequence to a plasmid, and induces transformation of the plasmid into a host cell capable of expressing this DNA sequence.

Prior to reacting the substrate in the presence of the enzymatic composition, the substrate may be modified so that its structure at the N-terminal side of C-terminal glycine has a molecular structure identical to at least one corresponding portion of the calcitonin sequence present in nature.

Calcitonin is preferably human or salmon calcitonin or analogs thereof. 1 »«

A method of producing a growth hormone releasing factor, which method comprises providing a substrate comprising an amino acid sequence corresponding to the amino acid sequence of at least one growth hormone releasing factor, wherein the substrate sequence f j | it has at least a glycine residue at its carboxyl terminus, and then the substrate is reacted in the presence of an alpha-amidating enzyme composition containing at least one

• · I

• *,: an alpha-amidating enzyme capable of catalyzing in the presence of oxygen and a reducing agent. ·: 25 at least at its C-terminus to the conversion of a peptidyl compound containing a glycine residue • · »! '. · A peptidyl amide having an amino group at the site of said C-terminal glycine, that '. * · The enzymatic composition is sufficiently pure in alpha amidating enzymes so that the specific activity of the enzymatic composition is at least about 25 mU / mg protein, · ·: preferably at least about 50 mU / mg protein and is sufficiently free from proteolytic acyl. * •; · '30 densities capable of degrading at least one entity consisting of a peptide: dyl compound, the corresponding peptidyl amide and alpha amidating enzymes so that the composition can catalyze the conversion when the peptidyl compound contains L-amino acids. Even more preferably, the specific activity of the enzymatic composition is at least about 150 mU / mg protein.

110433 67

The substrate may be obtained by a recombinant technique which isolates or prepares a DNA sequence capable of expressing the growth hormone releasing factor amino acid sequence, attaches the DNA sequence to a suitable plasmid, and induces transformation of the plasmid into a suitable host cell capable of expressing this DNA sequence.

Prior to reacting the substrate in the presence of the enzymatic composition, the substrate may be modified so that its structure at the N-terminal side of the C-terminal glycine has a molecular structure identical to that of at least one growth hormone releasing factor.

A method for preparing a calcitonin gene-related peptide, comprising providing a substrate comprising an amino acid sequence corresponding to at least one calcitonin gene-related peptide type, wherein the substrate 15 has a glycine residue at its carboxyl terminus and then reabsorbs the substituent. containing at least one alpha amidating enzyme capable of catalyzing the conversion of a peptidyl compound containing a glycine residue into an peptidyl amide having at least one C-terminal glycine in the presence of oxygen and a reducing agent at the C-terminus thereof, the specific activity of the enzymatic composition is at least about 25 mU / mg protein,: preferably at least about 50 mU / mg protein and is is free of proteolytic activity that can degrade at least one entity, which is a peptide »1 · V dyl compound, the corresponding peptidyl amide and alpha amidating enzymes so that the composition can catalyze the conversion when: the peptidyl compound contains L-amino acids. More preferably, the specific activity of the enzymatic composition is at least about 150 mU / mg protein.

• 1 *; ': The substrate may be obtained by a recombinant technique in which: •; · A DNA sequence capable of expressing the amino acid sequence ::: of a calcitonin gene-related peptide is inserted into a suitable plasmid and induced transformation of the plasmid :: to a suitable host cell capable of expressing this DNA sequence.

»· · * · · 110433 68

Prior to reacting the substrate in the presence of the enzymatic composition, the substrate may be modified so that its structure at the N-terminal side of C-terminal glycine has a molecular structure identical to that of at least one calcitonin related peptide.

16. A method for preparing alpha amidation enzyme compositions, comprising providing a DNA sequence capable of encoding an alpha amidation enzyme, inserting the DNA sequence into an expression vector, inducing transformation of the expression vector into a host cell capable of expressing the DNA sequence, and preferably selecting a DNA sequence. is this DNA sequence and express it.

In the method, at least a portion of said DNA sequence is a cDNA derived from polyadenylated RNA from total cellular RNA known to express an alpha-amidating enzyme and wherein the cDNA is selectively isolated after being identified by oligonucleotide probes specifically complementary to quenches capable of encoding the amino acid sequences present in the alpha-amidation enzyme. The DNA sequence has at least one of the following fragments (a) v v v v 50v

20 AATTCCGGTCTTTAAGAGGTTTAAAGAAACTACCAGATCATTTTCCAATG

V v v v lOOv

AATGCCTTGGTACCATTGGACCAGTCACCCCTCTTGATGCATCAGATTTT

• * »· · · v v v v 150v

GCGCTGGATATTCGCATGCCTGGGGTTACACCTAAAGAGTCTGACACATA

•, ·, v v v v 200v: 25 ctttctgcacgtccatgcgtctacct • · · (b) v v v v 50v aattccgtctcagtttctgtttctcttgcatcttctgcaattctgaggag • * * v v v v lOOv 30 gtgggtttgttctccactttgg · · ·. ttcgtggacttcgatgccagcctttttaactgaccgatgctccatttttt • »v v v v 200v • · * cggtcagggtgaacttccacacggtgttgtgtgtgcgctcgaagacgg • · | An immobilized alpha-amidating enzyme composition comprising at least one alpha-amidating enzyme composition capable of catalyzing the conversion of a peptidyl compound containing a glycine residue to at least one C-terminus of the peptidyl compound containing at least one C-terminus of the peptide. glycine, wherein the enzyme is covalently bonded directly or indirectly to a support which is sufficiently rigid, alone or in combination with other materials, to withstand a continuous flow of glycine-extended peptidyl substrate for more than 24 hours without substantially losing alpha-amidation activity -fa amidation enzymes such that the composition has a specific activity of at least about 25 10 mU / mg protein and is sufficiently free from proteolytic activity n at least one entity comprising a substrate compound, a corresponding peptidyl amide product, and an alpha amidating enzyme such that the composition may catalyze the alpha amidation reaction when the peptidyl compound contains L-amino acids.

Preferably, the enzyme is covalently bonded to a gel formed from a water-soluble copolymer of an α, ω-di-amine crosslinked acrylamide.

18. An immunoaffinity column for purifying impure samples of an alpha amidation enzyme, the column comprising an immunoaffinity resin containing an immobilized matrix which is insoluble in aqueous solution and does not degrade with an alpha amidation enzyme, glycine-extended peptidyl compounds or antibodies; it has functional groups capable of binding proteins and wherein the groups are / c: covalently bound to counterparts of monoclonal or polyclonal origin. 1 t for substances that selectively bind the alpha-amidating enzyme. Preferably, the functional group of the resin is activated with cyanogen bromide.

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Claims (4)

70 110433 1. En isolerad DNA sequence, kanknetecknad därav, att: A. DNA sequence coder for peptidyl-glycine-alpha-amiderande • monooxygen; och • V 25: · * B. DNA sequence kan hybridiser med ätminstone oligonucleotide probe: 3 ′ 5 Oligo- TTA CTC ACG GAC CCG TGG TAA CCG GGA CAC CAC TGG GGG GAC CTA CG nucleotide-T G • · probe 30 AE1 Iti 3 '5' Oligo- TTA CCG GTC ACC TG: Y: Nucleotide- G T T. *,! probe A * 14: v. 3 '5': · 35 Oligo- TTA CCG GTC ACC TG: Nucleotide- G A T probe AE5 v2 110433 3 '5' Oligo- TAC GTC GGI CCI AG I CTG GTC TT Nucleotide- T AT probe AE6 3 '5' 5 Oligo- TAC GTC GGI CCI TCA CTG GTC TT Nucleotide- T GAT Probe AE7 Oligo- 3 '5' Nucleotide- AC CCG TGG TAA CCG GGA CAC TG Probe IIIIII II AE8 10 3 '5' Oligo- AAA CAC TGC GTC ACC CCC CT nucleotide- GIITI probe AE9 3 '5' Oligo- CTG GTC TTG GTG AA nucleotide- AAA probe AE10 3 '5' Oligo- CTG GTT TTG GTG AA nucleotide- AAA probe AE11 An isolated DNA sequence, characterized in that 5 A. The DNA sequence encodes a peptidyl glycine alpha amidating monoxygenase; and B. The DNA sequence is hybridizable to at least two of the following oligonucleotide probes: 10 3 '5' Oligo-TTA CTC ACG GAC CCG TGG TAA CCG GGA CAC CAC TGG GGG GAC CTA CG Nucleotide TG Dikoetin ΆΕ13 3 '5' Oligo-TTA CCG GTC ACC TG Nucleotide-GTT Dikoetin AE4 3 '5' Oligo-TTA CCG GTC ACC TG Nucleotide-GAT Dikoetin AE5 3 '5' Oligo- TAC GTC GGI CCI AGI CTG GTC TT Nucleotide-T AT Dikoetin AE6 3 '5': Oligo - TAC GTC GGI CCI TCA CTG GTC TT Nucleotide- TGAT ·. '. * · Dikoetin ··, ·, AE7: Oligo- 3' 5 »: nucleotide- AC CCG TGG TAA CCG GGA CAC TG Dikoetin IIIIIIII • · 'AE8 • · · * * * 3 '5' Oligo- AAA CAC TGC GTC ACC CCC CT Nucleotide- GIITI; '· I Dikoetin * · "AE9. ··' 3 '5' Oligo- CTG GTC TTG GTG AA Nucleotide- A AA, ···, dikoetin AE10! '· *: 3' 5 '\ Oligo- CTG GTT TTG GTG AA: nucleotide A AA dikoetin AE11 110433 A prokaryotic or eukaryotic host cell, characterized in that it has been transformed or transfected with an expression vector comprising the DNA sequence of claim 1. 5 3. A method for recombinant expression of peptidyl-glycine-alpha-amidating monoxygenase (PAM), characterized in that the host cells of claim 2 are cultured for a sufficient period of time to obtain expression of PAM and then PAM is recovered. A process for the preparation of an amidated peptide comprising contacting a peptide having a C-terminal glycine with oxygen and a reducing agent in the presence of a peptidyl-glycine-alpha-amidating monoxygenase produced by the recombinant method of claim 15. 20 2. En prokaryotic eller eukaryotic disk, cellular transducer, att den and transformerad eller transfection med en expression vector, som innehäller DNA sequence: *: patentkravet 1. • · · · t · * 25 3. Förfarande för recombinant expression av peptidyl-glycin-j alpha-amiderande monooxygenase (PAM), kännetecknat därav, att I t t · jV. man odlar Värdceller enligt patentkravet 2 tillräckligt <· länge för att ästadkomma expression av PAM och därefter ätervinner PAM. 30 »* * • ·, ···, 4. Förfarande för framställning av en amiderad peptid, * · ·. ·, Kännetecknat därav, att man bringar en peptid, som • ·» i * · innehäller C-terminal glycin, i kontakt med syre samt ett reduktionsmedel in nerve av en peptidyl-glycine-alpha ", · 35 amiderande monooxygen, som framställts enligt *, * ·: recombinant förfarandet enligt patentkravet 3.