JPH074238B2 - Process for producing peptide C-terminal amidating enzyme - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a peptide C-terminal amidating enzyme, and more particularly to a method for producing the enzyme using a corresponding cDNA.

[Conventional technology]

An enzyme involved in the C-terminal amidation of a peptide C-terminal glycine adduct by an in vivo enzymatic reaction is called peptidylglycine-α-amidating monooxygenase (peptide C-terminal amidating enzyme) (EC.1.14.17.3). (Bradbury et al., Nature, 298 , 686,1982: Glembotski et al., J.
Biol. Chem., 259 , 6385, 1984), and is believed to catalyze the following reactions.

Utilization for elucidation of amidation mechanism in vivo, and method for converting peptides produced by recombinant DNA technology to physiologically active peptides, such as calcitonin and gastrin, only after C-terminal amidation. Therefore, attempts have been made to purify this enzyme, for example, bovine pituitary middle lobe (Murthy et al., J. Biol. Chem., 261 , 1815, 19).
86), pig pituitary gland (Kizer et al., Endocrinology. 118 , 226.
2,1986: Bradbury et al., Eur. J. Biochem., 169 , 579,1987),
Pig atrium (Kojima et al., J.Biochem., 105, 440,1989) , Xenopus body skin (Mizuno et al., Biochem.Biophys.Re
s.Commun., 137, 984,1986), rat thyroid tumor (Mehta
Et al., Arch. Biochem. Biophys., 261 , 44, 1988).

However, although it is possible to produce the above-mentioned C-terminal amidated peptide using these purified enzymes, it is premised that these are extracted from organism tissues and separated and purified. There are various difficulties in using sucrose in an industrial manufacturing process.

Therefore, in order to provide for the large-scale production of peptide C-terminal amidating enzyme using recombinant DNA technology, which has come into common use, isolation of corresponding cDNAs required for its expression and production of the enzyme using them have been attempted. ing. For example, Eipp
er BA et al., Mol. Endocrinol 1,777 ~ 790,1987 Ohsuy
e, K et al., Biochem.Biophys.Res.Commun.150, 1275-128.
1,1988, and Stoffers, DA et al. In Proc. Natl. Acad.
Sci. USA, 86,735 ~ 739,1989, respectively, the pituitary gland of cattle,
We have published a peptide C-terminal amidase cDNA derived from frog skin and rat atrium, and although it is not always satisfactory in its productivity, recombinant DNA technology using frog-derived and bovine-derived cDNA was developed. Production examples of the peptide C-terminal amidating enzyme used are also known (for example, JP-A-1-104168 and International Publication).
See WO89 / 02460 publication).

[Problems to be Solved by the Invention]

Although the above-mentioned technique using cDNA can be a starting point for mass production of peptide C-terminal amidating enzyme, it cannot be said that the mass production has been successful yet.

Therefore, the object of the present invention is to more efficiently use peptide C by using recombinant DNA technology that more effectively utilizes the corresponding cDNA.
It is intended to provide a means for producing a terminal amidating enzyme.

[Means for Solving the Problems]

The present inventors have also succeeded in isolating rat- and horse-derived peptide C-terminal amidating enzymes and preparing their cDNAs. It was surprisingly found that the use of the fragment excluding the region corresponding region not only secretes the produced enzyme outside the host cell but also significantly increases the overall production amount. Therefore, the above-mentioned object is to culture a host cell transformed with a plasmid comprising the peptide C-terminal amidase cDNA excluding the portion corresponding to the transmembrane region of the present invention, and capable of expressing the cDNA. Thus, the problem can be solved by a method for producing a peptide C-terminal amidating enzyme, which comprises collecting the enzyme or a precursor thereof from a culture in which the enzyme has been produced and accumulated.

Hereinafter, the present invention will be described more specifically.

C-terminal amidase cDNA that can be used in the present invention
Are humans, cows, horses, pigs, sheep, rabbits, goats,
The amino acid sequences of peptide C-terminal amidating enzymes present in mammals such as rat and mouse, birds such as chicken and turkey, amphibians such as frog, reptiles such as snake, fish such as sardines, mackerel, eel, salmon, etc. DNA encoding
The origin is not limited as long as it is derived from Escherichia coli and the DNA portion corresponding to the transmembrane region thereof is removed, but preferable examples include those derived from mammals. More specifically, the amino acid sequences of the currently known peptide C-terminal amidating enzymes are represented by single-letter amino acids, and a missing portion (indicated by-) is arbitrarily added to increase homology between species. A cDNA fragment which is inserted and encodes the amino acid sequence as shown in FIG. 1 and in which the portion corresponding to the hydrophobic amino acid region near the C-terminus thereof is removed can be advantageously used. In addition, each cDNA is equine, bovine, rat, frog I
And Frog II respectively; Mol. Endocrinal.
1 , 777-790 pages, 1987; Proc.Natl.Acad.Sci.USA, 86 ,
735-739, 1989; Biochem.Biophys.Res.Commum., 1
48 , pp. 546-552, 1987; and Biochem. Biophys. Res.
Commum., 150 , pp. 1275-1281, 1988. Among these, for example, according to the sequences of horses and rats in FIG.
The region up to the Ith (isoleucine) corresponds to the hydrophobic amino acid region. Therefore, the transmembrane region in the present invention refers to the hydrophobic amino acid region of the target cDNA.
The peptide C-terminal amidating enzyme cDNA excluding the portion corresponding to such a region may be prepared by excising the portion from the known cDNA using a restriction enzyme known per se, or the cDNA of the cDNA. It is also possible to select from various cDNAs produced by differences in mRNA splicing at the cloning stage. For example, the cloning of the cDNA used in the present invention can be carried out by a method known per se using various tissues of various animals described above. In particular,
+,-Methods, hybridization methods, PCR methods and other commonly used methods (eg, Methods in Enzymolog
y, vol.152; Guide to Molecular Cloning Techniques, S.
L. Berger and ARKimmel, 1987, Academic Press, IN
C.; Methods in Molecular Biology, vol.4; New Nucleic
Acid Techniques, JM Walker, 1988, The Humana Pres
s Inc.; Molecular Cloning A Laboratory Manual 2nd E
dJSambrook, EFFritsch, T. Maniatis, 1989, Cold S
pring Harbor Laboratory press)
The cDNA region encoding the protein is determined by determining the nucleotide sequence of the obtained cDNA clone, and it may be selected from those in which the portion corresponding to the above-mentioned transmembrane region has been removed.

In the case of a rat, for example, a tissue that produces a large amount of peptide C-terminal amidating enzyme, for example, a rat pituitary is homogenized with guanidyl thiocyanate to disrupt the cells, and cesium chloride equilibrium density gradient ultracentrifugation is used to separate RNA. Get fractions. Then, by affinity chromatography supporting oligo dT cellulose, the RN
RNA with poly A (poly A ⁺ RNA) is isolated from the A fraction.

Using this poly A ⁺ RNA as a template, a known method, preferably the method of Okayama-Berg (Mol.Cell.Biol.2,161,1982)
To obtain a cDNA library. Positive clones were screened from these libraries using appropriate probes, and positive cDNA clones obtained by rescreening using amplified probes from the amplified cDNA libraries were isolated, and their restriction enzymes were isolated. Target cD such as by mapping and sequencing
NA can be structurally determined. In addition, a plasmid containing the desired cDNA can also be selected by incorporating the cDNA into an expression vector and evaluating the productivity of the peptide C-terminal amidase of the host transformed with this expression vector.

A host for expressing this cDNA may be a commonly used cell such as microorganisms such as Escherichia coli, Bacillus subtilis, yeast, cultured cell lines derived from insects, animals and the like. If the expression plasmid is a plasmid that can efficiently express cDNA in these cells,
anything is fine. For example, it can be appropriately selected from those described in the following books.

Continuation Biochemistry Experiment Course 1, Gene Research Method II, One Recombinant DNA Technology-Chapter 7, Expression of Recombinant, (1986), edited by The Japanese Biochemical Society, Tokyo Kagaku Dojin; Recombinant DNA, Part D, Section I
I, Vectors for Expression of Cloned Genes, (1987) R
ayWu and Lawrence Grossman, Academic Press, IN
C.; Molecular Cloning, A Laboratory Manual 2nd Ed.Bo
ok3, (1989) J. Sambrook, EFFritsch and T. Maniatis.
Edited by Cold Spring Harbor Laboratory Press.

For example, when CV-1 which is commonly used as animal cultured cells is used as a host, a pSV, pL2n, pCol type promoter and optionally a selection marker may be used. Further, pGH, pKYP, pHUB type vectors can be used for Escherichia coli, and YRp, YEp type vectors can be used for yeast. Recombination of these vectors with cDNA, and transformation and transduction of host cells with recombinant plasmids can be carried out by procedures known per se described in the aforementioned references and the like. The transformed cells thus obtained can be cultured in the medium and culture conditions usually used for growing the cells from which they are derived.

The peptide C-terminal amidating enzyme produced and accumulated from such a culture can be easily collected from the culture medium after the cells have been removed, since the produced enzyme is secreted extracellularly, for example, when using animal cultured cells. However, it may be collected from a cell lysate if necessary. This collection / purification can be carried out by combining an ordinary enzyme purification method, for example, fractionation by precipitation, heparin affinity chromatography, dialysis and the like. Furthermore, the C-terminal glycine adduct developed by the present inventors is used as a ligand. It is preferable to use in combination with the substrate affinity chromatography described above (see International Publication WO89 / 12096). Peptides consisting of 2 to 6 amino acid residues including glycine, especially D-Tyr-Trp-Gly, are used as ligands for this chromatography.
Those using Phe-Gly-Phe-Gly and Gly-Phe-Gly are preferred. A specific example of the purification procedure can be performed according to the description in the above publication.

〔Example〕

The following example describes the production of the enzyme using the peptide C-terminal amidase cDNA derived from rat pituitary, but the present invention is not limited thereto.

Example 1. Preparation of expression plasmid When cDNA cloning was performed using rat pituitary-derived poly A ⁺ RNA, 5 cDNAs having different molecular weights were obtained (Fig. 2, Fig. 3, biochemistry, 62,842 (1989)). Cleaved with EcoRI- XmaI of cDNA clone 202 2.
The 58 kbp (kilobase pair) DNA fragment was used as an animal culture cell line expression vector pSV2 vector [S. Subramani, R. Mulligan,
P. Berg, Mol. Cell. Biol. 1 , 854 (1981)] Hin d III- B.
This plasmid was named SV-205 by inserting it into the gl II site via a synthetic linker. Next, SV-205 Nsi I (7
The 00) -XmaI fragment was replaced with the NsiI (700) -XmaI fragment of each of the cDNA clones 201, 202, 203, 204 . These expression plasmids were designated as SV-201, SV-202, SV-203 and SV-204. Of these, it was confirmed that SV-203 had a portion corresponding to the transmembrane region of cDNA removed. Therefore, SV-20
3 is the plasmid according to the present invention, and the others are comparative examples.

Example 2. Expression in cultured animal cells Cultured cells COS-7 were cultured in a synthetic medium containing 10% fetal bovine serum (D
MEM) and transformed with the expression plasmid of Example 1 by a known method (C. Chen and H. Okayama, Mo.
l, Cell. Biol. 7 , 2745 (1987)). At this time, 20 μg of expression plasmid was used for 5 × 10 ⁵ cells. After culturing for 24 hours under conditions of 3% carbon dioxide and 35 ° C, 10 ml of DMEM medium containing 0.2% bovine serum albumin (BSA)
After washing the cells twice with 10 ml of DMEM medium containing 0.2% BSA
The medium was further cultured under the conditions of 5% carbon dioxide and 37 ° C. for 48 hours.

Example 3. C-Terminal Amidating Enzyme Activity Produced by Recombinant Cells The cell culture medium expressed in Example 2 was separated into cells and supernatant (medium) by centrifugation.

The enzyme activity of the supernatant was measured. The measurement of amidating enzyme activity is performed in the literature (Tohoku J. Exp.Med. 156 , 191 (1988)).
The method described in 1. was followed. That is, 40 picomoles of D-tyrosyl-L-valylglycine and ¹²⁵ I-D-tyrosyl-L-valylglycine were used as reaction substrates,
The reaction was carried out at 37 ° C. for 5 to 10 hours in a reaction solution containing 100 μg / ml catalase and 1 mM ascorbic acid 50 μM copper ions in 50 mM Hepes-potassium buffer having a pH of 8.5. The enzyme activity was measured by separating ¹²⁵ I-D-tyrosyl-L-valine amide and D-tyrosyl-L-valine amide produced by using an ion exchange column and measuring the radioactivity thereof with an r-counter.

The measurement results are shown in Table 1.

The peptide C-terminal enzyme produced by the animal cultured cells using the plasmid SV-203 according to the present invention is about 5 times or more of the enzyme level in Comparative Example, and the transmembrane region portion of the corresponding cDNA is removed. It can be seen that there is a significant increase.

〔The invention's effect〕

According to the present invention, the peptide C-terminal amidase can be produced extremely efficiently in a specific recombinant plasmid-host system of the corresponding cDNA.

[Brief description of drawings]

FIG. 1 shows the amino acid sequence deduced from the peptide C-terminal amidase cDNA cloned from horse, cow, rat and frog in single-letter code. FIG. 2 shows the nucleotide sequence of C-terminal amidase cDNA cloned from rat pituitary mRNA and the amino acid sequence deduced therefrom. FIG. 3 schematically shows five C-terminal amidase cDNA clones cloned from rat pituitary mRNA. The region encoding the putative enzyme is indicated by a box. Numbers indicate the number of bases (bp) with the translation start point as 1. TM indicates a portion corresponding to the transmembrane region. The restriction enzymes are indicated by the following abbreviations. B ( Bam H I), N ( Nsi I), RI ( Eco R I), RV ( Eco R V),
The nucleotide sequences of S ( Sph I) and X ( Xma I) regions A to C are shown in FIG.

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Claims (2)

[Claims] 1. A portion corresponding to a transmembrane region, comprising 880
By culturing a host cell transformed with a plasmid comprising a rat C-terminal amidase cDNA derived from rat, excluding the DNA sequence part encoding the amino acid region of -901, and capable of expressing the cDNA. A method for producing a peptide C-terminal amidating enzyme, which comprises collecting the enzyme or a precursor thereof from a culture in which the enzyme has been produced and accumulated. 2. The method according to claim 1, wherein the host cell is an animal cultured cell.