RU2120475C1 - Method of synthesis of polypeptide, hybrid dna (variants), fusion protein (variants) - Google Patents

Abstract

FIELD: biotechnology, recombinant polypeptides. SUBSTANCE: method involves making hybrid DNA - a structure encoding the protein in which two polypeptide sequences are bound by transient region containing site recognized by Ig A-protease. The host cells are transformed with recombinant expression vector containing the indicated structure, isolation of the obtained fusion protein, its treatment with Ig A-protease at weight ratio of enzyme to substrate from 1: 1 to 100:1 at pH 6.5-8.5 and isolation of peptide. Specific forms of hybrid DNAs and fusion proteins are obtained, characterized and tested. Invention provides preparing methionineless polypeptides. EFFECT: improved method of synthesis, increased expression yield and purification degree of proteins. 19 cl, 11 ex, 4 dwg

Description

 The invention relates to a method for sequentially specific cleavage of proteins obtained by biotechnology using IgA proteases (also called gases), and especially to a method for producing recombinant proteins or peptides in prokaryotes and subsequent removal of the N-terminal sequence.

 Obtaining proteins by biotechnology is carried out using microorganisms that are easily cultivated and allow a simple way to obtain protein. Suitable microorganisms are, for example, the gram-negative bacterium Escherichia coli, the gram-positive bacterium Streptococcus carnosus and the baker's yeast Saccharomyces cerevisiae. However, the expression of authentic foreign genes in such microorganisms often has disadvantages. For example, in E. coli, the translation of the amino-terminal residue of the methionine of the natural proteins of the prosthesis is effectively cleaved in most cases. However, with foreign proteins, this cleavage of the first methionine residue occurs in most cases in parallel. Therefore, a suitable method is to obtain such proteins with a specific amino terminus, their formation first in the form of fused proteins and then their cleavage by means of a specific sequentially specific protease.

 In addition, in contrast to authentic proteins, such fused proteins have the advantage of being enlarged in the cell of the microorganism and forming dense precipitated bodies ("Inclusion Bodies"), which can be separated with little effort from other parts of the cell and thereby facilitate the isolation and purification of the desired protein. On the other hand, carrier proteins, which, using genetic engineering methods, primarily merge with the desired protein itself, give the merged partner special resistance against non-specific proteolytic cleavage; the problem of cleavage of polypeptides that are considered foreign, relates especially to the biotechnological production of smaller peptides. Other carrier proteins again allow the distribution of the desired proteins in specific cell compartments from which they can be especially easily purified, where they are particularly stable and / or where they are available for testing purposes. Finally, carrier proteins may also have special properties that allow for efficient purification, for example by affinity chromatography. For many uses, fusion proteins that carry a carrier protein at the amino terminus and a desired protein at the carboxyl end are preferred. But in certain cases, the inverse or binding of the desired protein to two fusion partners may also be desirable. Further, it may be advantageous to reverse the desired protein within the fusion.

 To obtain the desired protein in free form from such a fusion, cleavage of the covalently linked fusion partners from one another is necessary. In principle, this can be achieved using chemical or biochemical (enzymatic) methods. However, in most cases there is a limited specificity of the methods available so far; since in order to obtain the desired protein it is important that such cleavage occurs in the sequence between the fusion partners, i.e. in the transition region, but in no case additionally inside the desired protein itself. Therefore, the cleavage of the merger partners must be highly specific.

 The chemical methods used so far for sequentially specific separation of fused proteins are, for example, cleavage with bromine cyanide on the amino acid methionine inside the protein and cleavage between the amino acids Asp.'Pro in an acidic medium using formic acid. But these methods are only suitable if the specific cleavage site in the desired protein, regardless of the transition region to the fusion partner, is not repeated a second time. However, in general, biochemical methods of cleavage should be preferred to chemical methods, since the former can in most cases be carried out under physiological or at least chemically mild reaction conditions that do not prejudice the desired protein.

 Biochemical methods for cleaving fused proteins are based on the use of specific proteases if possible. For example, trypsin cleaves peptide bonds following the amino acids arginine or lysine within proteins. An increase in specificity can be achieved by the preceding chemical modification of the LYS amino acid, as a result of which specific recognition can be limited to the amino acid arginine. Another protease used in biotechnology is Clostripain; this enzyme cleaves peptide bonds between the amino acid arginine and any amino acid that follows it. A review of the enzymatic methods used so far to break down fused proteins was compiled by F.A. About Marston (B [D.M. Clover, E.]: DNA cloning III, JRL PRESS Oxford and Washington DC, 1987). Enzymatic cleavage methods are also limited as a result of the fact that the cleavage point-specific amino acid (s) can be found simultaneously also in the most desirable protein. Therefore, enzymes are especially suitable for the biochemical cleavage of protein fusions, which for cleavage recognize not only an amino acid, but also a sequence of amino acids; since the probability that a certain amino acid sequence other than at the cleavage point between the fusion partners occurs again in the most desirable protein, the smaller the greater the number of amino acids needed to recognize and cleave the cleavage sequence.

 Proteases that very specifically cut a specific protein are known. Although most of these selective proteases (which are found, for example, in the complementary system and in the human blood coagulation system) cleaves at a certain point in the substrate; however, when the corresponding cleavage region is transferred to another protein (for example, a fused protein), such proteases, as a rule, are no longer able to cleave. The reasons for this are varied and include, for example, recognition of a specific secondary or tertiary structure in the substrate by a protease or inaccessibility of the cleavage point in fused proteins.

The list of sequentially specific proteases that are still used to break down fused proteins currently contains factor X _a . This protease cuts the specific cleavage sequence of Ile-Glu-Gly-Arg X, moreover. . shows the cleavage point and X shows any amino acid. However, it turns out that this protease cannot be used universally for the cleavage of fused proteins that carry the corresponding cleavage sequence in their transition region; often, such substrates (i.e., fused proteins that contain the desired protein covalently linked to a carrier protein) do not cleave at all, cleave to a very small extent or only in soluble form.

 Particularly significant is the effective cleavage of fused proteins in the production of recombinant proteins in prokarion organisms. Namely, it requires the cloning of a DNA sequence that contains AUG as an initial codon before starting its own DNA sequence. As a result of this, a recombinant protein that contains a methionine residue at position-1 of the amino acid is expressed in prokaryonts, such as, for example, E. coli.

 However, in many cases, production of recombinant proteins that do not contain methionine at position-1 is required. The production of such proteins from prokaryonts can be carried out, for example, through methionine-specific peptidase, which cleaves the N-terminal methionine. However, this method is associated with very high costs, since cleavage can only be verified through a sequence of proteins. In addition, the separation of the methionine-containing at position-1 and the methionine-free protein due to the almost identical molecular weight is very difficult, and thus can only be achieved incompletely.

 PCT application W084 / 02351 discloses the cleavage of N-terminal amino acids from a fused protein. Moreover, many amino acids of the protein from the N-terminus due to the gradual cleavage of exopeptidase, preferably leucine peptidase, can be removed to the sequence X - Pro. The X - Pro sequence is cleaved from this product in either two steps or 1 step reaction using postproline dipeptidyl aminopeptidase (EC 3.4.14). However, this method has the disadvantage that due to the gradual cleavage of amino acids of the N-terminus of the protein, not a single product should be formed, but always a mixture of products, which, along with the desired product, contains both incompletely and a significant amount of cleaved proteins.

 Another method for the enzymatic cleavage of a fused protein is known from European patent N 0020290. In this method, the Collagenase enzyme is used to cleave a fused portion of a protein with a specific recognition sequence. After this, other amino acids of the fused portion can then be removed as a result of further enzymatic treatment. However, it was found that Collagenase and also other endopeptidases have only a small specificity (see Biochim Biophys Acta 271 (1972), 133-144). In addition, Collagenases are active only with proteins that have a specific spatial structure.

The use of the aforementioned factor X _a for cleaving the N-terminal portion of a protein fusion is known. However, this method, along with the already mentioned problems of cleavage efficiency, has other disadvantages, namely of the type that, to the extent possible, internal sequences of the protein are recognized and cleaved. In addition, factor X _a should be isolated from the blood serum of cattle, as a result of which cleavage of the proteins used in therapy requires cost-related purification and analysis in order to detect possible pathogenicity factors or viruses.

 Different types of pathogenic bacteria (for example, the genus Neissria, such as Neisseria gonorrhoea and Neissria meningititis, or the genus Haemophilus, like Haemophilus influenrae and Haemophilus aegypticus, which develop on the human mucosa, are distinguished by their sequence close to related proteases that are specific for human IgAl and therefore, in sum, they are referred to as IgA proteases or igases. IgAl immunoglobulin is an important component of the secret immune response, which should protect against infections of such pathogenic organisms (Review: Jornfeld and Plaut, Rev, Jnfect. Dis 3 (1981), 521-534) Along with this, the IgA protease also cleaves its own precursor protein as a result of autoproteolysis.The formation and action of the IgA protease from Neisseria gonorrhoeal MSll in an authentic bacterial strain and in gram-negative host cells has already been described in detail (German Federal Patent No. 3622221.6 EP 0254090).

IgA protease cleaves the following recognition sequences, as, for example, described by Pohlner (Nature 325) (1987), 458-462):
1.Pro - Ala - Pro 'Ser - Pro
2. Pro - Pro. '. Se - Pro
3. Pro - Pro. '. Ala - Pro
4. Pro - Pro. '. Thr - pro
With this symbol. '. denotes, respectively, the cut point of the IgA protease. Cloning of an IgA protease is described above, for example, by Pohlner et al., 1987.

 Therefore, the objective of the present invention was to develop an improved method for biochemical (enzymatic) cleavage of fused proteins, so that the fused proteins obtained by the method of genetic technology, consisting of any fusion partners and a specific sequence of cleavage in the transition region, can be used as a substrate to obtain the desired proteins, if possible high output and reproducibility.

 This problem was solved by genetic technology by introducing a recognition point or a Pro'X - Pro cleavage sequence into the transition region of fused proteins and by specifically cleaving this IgA protease cleavage sequence at the designated symbol. ' a cleavage point, wherein X denotes primarily the amino acids Ser, Thr Ala, and particularly preferably Ser or Thr, but may also denote other amino acids.

Therefore, the subject of the present invention is a method for enzymatically cleaving fused proteins and for obtaining the desired parts of these fused proteins, which is characterized in that
(1) the transition region in which two parts of the fused protein are linked to each other is modified by genetic engineering such that at least one IgA protease recognition point with the amino acid sequence Y - Pro is formed in this transition region. '. X is Pro, wherein X may be any amino acid and Y one or more amino acids,
(2) the fused protein obtained from step (1) is cleaved with an IgA protease at the designated symbol. '. recognition point position and
(3) after cleavage, one part or more of the desired parts of the fused protein is obtained.

 The term “IgA proteases” of the present invention includes proteases that specifically cleave IgA, which are described, for example, in Rev. Infekt. Dis. 3 (1981 521-534). But also suitable are recombinant IgA proteases, which are described, for example, in the patent application of Germany N 3622221, Proc. Natl. Acad Sci USA 79 (1982) 7881-7885, Proc. Natl. Acad Sci U.S. 80 (1983) 2681-2685, Nature 325 (1987) 458-462 and EMBO Jour 3 (1984) 1595-1601.

 In the method according to the invention, the transition region of the fused proteins is modified predominantly in such a way that nucleotide sequences that encode the IgA protease recognition point or part of it are inserted into the transition region of the fused protein, and these nucleotide sequences are inserted in front of and / or behind one or more of the coding ones parts of the fusion of DNA proteins - segments. For this purpose, mainly nucleotide sequences that have been synthesized chemically are used.

 It has surprisingly been found that the method of the invention is also particularly suitable for cleaving fused proteins that initially (i.e., before modifying the transition region) do not have any natural recognition point for the Iga protease.

 The IgA protease recognition point for the method of the invention has a consistent amino acid sequence of Y - Pro. '. X - Pro. Moreover, X may denote any amino acid and Y may denote one or more of any amino acids. Preferably, X is serine, threonine or alanine, especially predominantly serine or threonine. Y denotes predominantly many amino acids that end with the sequence Pro, Pro — Ala, Arg — Pro, Pro — Arg — Pro, Ala — Pro — Arg — Pro, or Pro — Ala — Pro — Arg — Pro.

 Therefore, the method of the invention discloses that an IgA protease recognition point is at least with a coordinated Pro cleavage sequence. '. X - Pro can be introduced into the transition region of any fused protein, for example, between the carrier protein and the desired protein, and used to cleave and produce the desired protein through IgA protease. Moreover, in the cleavage sequence Pro. '. X - Pro mainly use the amino acids Ser, Ala or Thr at position X. To further optimize the cleavage at this point, you can include other special amino acids, especially the amino acid Pro, in the cleavage sequence.

Especially preferred amino acid sequence
a) Pro - Ala - Pro. '. Ses - Pro
b) Pro - Pro. '. Ser - Pro,
c) Pro - Arg - Pro - Pro. '. Ala - Pro,
d) Pro - Pro. '. Thr - Pro,
e) AlA - Pro - Arg - Pro - Pro. '. Thr - Pro or
f) Pro - AlA - Pro - Arg - Pro - Pro. '. Thr - Pro.

 When applying the method of the invention to cleavage of fusion proteins in which the desired protein is included after the carrier protein, a protein is formed after cleavage by the IgA protease, the amino terminus of which is characterized by the sequence X - Pro. This sequence, as part of the desired protein, may be beneficial, disadvantageous, or negligible. This sequence is advantageous, in general, when the desired protein obtained by the method of genetic technology and in its natural form contains both corresponding amino acids X - Pro on its amino terminus. The amino-terminal X - Pro characterized proteins, which are important in biotechnology, are found in nature.

 The method according to the invention, in contrast to all other known methods for splitting protein fusions, has the advantages that it can unexpectedly be applied universally to fused proteins that carry the indicated cleavage sequence in their transition region and that it can also be applied to insoluble, soluble, joined membrane and cell-bound protein fusions. In addition, as a particular advantage, the method makes it possible to break down fused proteins or protein fusions in the form of precipitated bodies of the kind that they are obtained in microorganisms and where they are easily enriched as such. Another advantage of the method is that the cleavage enzyme used, Igase, can be obtained at low cost from nutrient media of non-pathogenic bacteria.

 The insertion of the cleavage sequence for Jgas in the transition region of protein fusion is carried out by means of gene technology. For example, a nucleotide sequence or a nucleotide sequence that encodes a cleavage sequence or part of it can be chemically synthesized and inserted using known genetic technology between DNA segments for a carrier protein and a desired protein. Accordingly, you can also embed a natural nucleotide sequence that encodes a suitable cleavage sequence or part of it. The gene encoding the fusion of proteins is placed primarily under the control of appropriate (predominantly inducible) expression signals, so that appropriate production of fused proteins becomes possible. As host cells for the production of protein fusions, prokaryon or eukaryon (both plant and animal) cells can be used; but cell-free systems are also possible. The carrier proteins used for this can have any function, depending on what properties they should give to protein fusion, as certain transfer functions, functions that improve the purification of protein fusion or their stability, and much more. Preferred carrier proteins are explained below.

 The protein fusion cleavage according to the method according to the invention is mainly carried out using Jgas, which is formed from a super-producing non-pathogenic strain of bacteria and obtained by purification from supernatant liquids of a culture (see, for example, German Federal Patent No. 3622221).

 The method according to the invention can be used both for production and for analytical purposes. The method serves to obtain important proteins by the biotechnology method, which can be used, for example, in medicine, in scientific research, in environmental protection, or in industrial processes or products. For analytical application, the method in combination with suitable expression systems can serve, for example, for a standard study of gene fusions.

A preferred embodiment of the method of the invention for enzymatically cleaving fused proteins and for preparing desired portions of these fused proteins is characterized in that
(1) transforming a cell with recombinant DNA or with a recombinant carrier, the DNA or carrier containing at least a copy of a gene encoding a fused protein that contains at least a transition point for the recognition of an IgA protease,
(2) cultivating the transformed cell in a suitable medium,
(3) adjusting the expression of the fused protein encoding gene in the transformed cell,
(4) cleave the fused protein with an IgA protease
(5) isolate one or more desired parts of the fused protein.

 In this case, the treatment of the fused protein with an IgA protease in a medium (nutrient broth) can be carried out after dissolution of the cells and / or after partial or complete separation of the cellular proteins.

 Further, it is preferable for the treatment of the fused protein, mainly the prokaryon expression product, to immobilize the IgA protease by a method known to the person skilled in the art, for example as described in European Patent B 0141223 or European Patent B 0141224.

A particularly preferred application of the method of the invention is to produce recombinant proteins or peptides without the N-terminal methionine residue from fused proteins or peptides with the amino acid sequence Met-Y-Pro. ' X is Pro — A, where X is any amino acid, preferably Thr, Ala or Ser, Y is one or more amino acids, which preferably, if X is Thr or Ala, ends with Pro, or if X is Ser, ends with the sequence Pro - Ala or Pro is Pro, and A denotes any amino acid sequence. In this case, the fused protein or peptide is cleaved using an Iga-protease and a cleavage product with the amino acid sequence X - Pro - A is obtained. For example, this method for producing recombinant proteins from prokarion cells without an N-terminal methionine residue includes the following steps:
(1) transformation of a prokaryon cell with a gene that encodes a protein or peptide with a Met - Y - Pro amino acid sequence. '. X - Pro - A, where X, Y and A have the above meanings.

(2) culturing the transformed cell in a suitable medium and the expression of the transformed gene,
(3) cleavage of the expression product from the transformed cell with the amino acid sequence Met - Y - Pro. ' . X - Pro - A IgA protease and
(4) isolation of the resulting cleavage product with the amino acid sequence X - Pro - A without the N-terminal methionine residue.

 Thanks to the method of the invention, it is surprisingly possible to obtain proteins with no N-terminal methionine residue with high N yield and good specificity in one step, which have an N-terminal X-Pro sequence, wherein X is predominantly Thr, Ala or Ser.

 Part of the carrier Y of the fused protein is an amino acid sequence of at least 1, preferably up to 100, especially preferably from 1 to 50 amino acids, which ends with a recognized IgA protease cleavage sequence. If X represents the amino acid serine, then Y ends predominantly with the sequence Pro — AlA or Pro. If X stands for Thr or Ala, then Y ends predominantly Pro, especially predominantly Arg - Aro, Pro - Arg - Pro or Ala - Pro - Arg - Pro.

 In a particularly preferred embodiment, Y is at least 5 amino acids that end with the sequence Pro — Ala — Pro — Arg — Pro. However, for the method according to the invention, all recognized IgA - protease cleavage points are used.

 Part of the carrier Y may contain any other amino acids, preferably up to 100, especially preferably up to 50 amino acids. However, for this, predominantly amino acid sequences are used which, at the DNA level, improve the expression of the Met-Y-Pro protein. ' X - Pro - A or / and at the amino acid level facilitate its purification from the cell.

 The expression of protein Met - Y-Pro. '. X - Pro-A at the DNA level can be improved, for example, by fusion with fragments of the β-galactosidase gene, i.e. part of the carrier Y includes part of the β-galactosidase protein. Other possibilities are known to those skilled in the art to increase the expression of the Met-Y-Pro protein. '. X - Pro - A. By fusion with other polypeptides, especially with highly charged (e.g. poly (Lys, Arg)) or with binding substances with high affinity (e.g. streptavidin) polypeptides or proteins, it is possible to facilitate the purification and separation of the expression product (see for example, European patent A 0089626, European patent A 0306610).

Further, the subject of the present invention is a fusion protein that contains many parts of the polypeptide and which, inserted at least in the transition region between the different parts of the polypeptide, has one or more IgA protease recognition points with the amino acid sequence Pro. '. X is Pro, wherein X is any amino acid, but preferably Ser, Thr or Ala. Particularly preferably, the recognition point has an amino acid sequence
(a) Pro - Ala - Pro. '. Ser - Pro,
(b) Pro - Pro. '. Ser - Pro,
(c) Pro - Arg - Pro - Pro. '. Ala - Pro,
(d) Pro - Pro. '. The - Pro,
(e) Ala - Pro - Arg -Pro -Pro. '. Thr - Pro or
(f) Pro - Ala - Pro - Arg -Pro - Pro. '. The - Pro,
moreover. '. shows the splitting point.

 The present invention also includes, in particular, a protein or peptide with a sequence of amino acids Met-Y-Pro. '. X is Pro — A, where X is predominantly Thr, Ala or Ser, Y is one or more of any amino acids, and preferably, if X is The or Ala, it ends with Pro, or if X is Ser, it ends with the sequence Pro — Ala or Pro and A denotes any amino acid sequence. Such a protein or peptide is expressed by the method of the invention by transforming a prokaryon cell with a recombinant carrier that contains at least a copy of a gene that encodes a similar protein or peptide.

 Sequence A may denote any amino acid sequence. However, it is advisable that there is no other cleavage point for the IgA protease within this amino acid sequence.

 Further, the subject of the present invention is a recombinant DNA that colorants a protein or peptide of the invention and where at least one recognition point of an Iga protease or cleavage sequence is inserted in at least the transition region of the fused protein.

 The recombinant DNA according to the invention can be obtained by a method known to the person skilled in the field of molecular biology. Typically, for this, a carrier that contains the coding sequence of amino acids A of the DNA sequence is cleaved with restrictive endonucleases at the 5 ′ end of this gene and re-ligated with oligonucleotides that contain the desired sequence. Moreover, the oligonucleotide must contain a sequence that encodes a cleavage point of an IgA protease or part of it.

 Further, the subject of the invention is also a recombinant carrier that contains at least a copy of the recombinant DNA of the invention. Suitable carriers for suitable protein expression in prokaryotic organisms are known to those skilled in the art. A suitable carrier is one which provides high expression of the recombinant DNA of the invention. Recombinant DNA is predominantly controlled by an inducible expression signal (e.g., λ, tac, Iac, or trp promoter).

 The carrier of the invention can be either extrachromosomal (e.g., plasmids), or integrated (e.g., bacteriological lambda) in the genome of the host organism.

 Advantageously, the carrier of the invention is a plasmid. Carriers that are suitable, respectively, for gene expression in a particular host organism, are known to those skilled in the field of molecular biology. It may be a eukaryon carrier, but mainly a prokaryon carrier. Examples of carriers suitable for expression of the DNA of the invention in prokaryons are commercially available pUC and puR carriers.

 Another subject of the invention is a cell, preferably a prokaryon cell, especially a E. coli cell, which is transformed with the recombinant DNA of the invention and / or with the recombinant carrier of the invention.

 Examples of proteins that have an N - terminal sequence X - Pro, where X is Thr, Ala or Ser, and which can be obtained by the method of the invention in one stage, are human erythropoietin, β - chain of the human T-cell receptor and especially granulocyte stimulating human factor (G - CSF).

 G - CSF is synthesized as a lymphokine of activated monocytes, macrophages, as well as a number of other cell lines. Lymphokines are involved in the maturation of cells of the immune system or blood cell system.

 They stimulate the maturation of major bone marrow cells to differentiated cells. So, for example, G - CSF induces the formation of neutrophylene and granulocytes.

 Since G - CSF can significantly increase the population of neutrophilic cells in a short period of time, there are wide application possibilities in the field of therapy for G - CSF. So, G - CSF could be used, for example, after chemotherapy for cancer, in which cells of the immune system are destroyed. Further, G - CSF could be used for bone marrow transplants, for severe burn wounds, for hostile infections caused by weak immunity, and for leukemia.

 G - CSF is a sector protein molecule. Therefore, the primary transplant product contains an N-terminal signal sequence that is cleaved during secretion, so that the sequence of mature G-CSF begins with amino acids Thr (+1) - Pro (+2) (amino acid positions +1 and +2). In the production of G-CSF in prokaryons, this signal peptide is only poorly cleaved or not cleaved at all, so to obtain G-CSF without a signal sequence from prokaryons, it is necessary to clone AUC (Met) as an initiating codon before starting the mature G-CSF coding DNA sequence, which starts at the protein level with Thr (+1) - Pro (+2). As a result, G - CSF, which contains methionine at the 1-position of the amino acid, is expressed in prokaryonts such as E. coli.

 According to the method of the invention, it is possible in a simple way to obtain the methionine-free amino acid in the 1-position of the amino acid G - CSF from prokaryonts, which begins with amino acids Thr (+1) - Pro (+2).

 This is due to the fact that the derivative G - CSF of non-prokaryonts is obtained, which at the positions +1 and +2 of the amino acid sequence contains the amino acids Thr (+1) - Pro (+2) and before that, starting from position - 1 of the amino acid sequence, contains an amino acid sequence that is recognized by the IgA protease and can be cleaved from the amino acid sequence G - CSF, which begins with Thr (+1) - Pro (+2).

 In a preferred embodiment, the derivative contains, at position -1 and -2 with respect to Pro, at position -3 up to position -I, the amino acid sequence Arg-Pro-Pro, at position 4- up to -1 the amino acid sequence Pro - Arg - Pro - Pro or position -5 to position -1 amino acid sequence Ala - Pro - Arg - Pro - Pro.

Под G - CSF в смысле изобретения понимают, конечно, существующий G - CSF, последовательность которого раскрыта, например, в Science 232 (1986) 61, а также образованные от него производные со стимулирующей гранулоциты активностью, у которых последовательности аминокислот начинаются с X (+1) - Pro (+2). X обозначает Thr, Ser или Ala, особенно предпочтительно Thr.In a particularly preferred embodiment, the derivative contains, at position -6 to position -1, an amino acid sequence

By G-CSF in the sense of the invention is meant, of course, the existing G-CSF, the sequence of which is disclosed, for example, in Science 232 (1986) 61, as well as derivatives derived from it with granulocyte-stimulating activity, in which amino acid sequences begin with X (+ 1) - Pro (+2). X is Thr, Ser or Ala, particularly preferably Thr.

 G-CSF derivative according to the invention after expression in prokaryons can be cleaved by treatment with an IgA protease between position +1 and -1 (between Thr (+1) and Pro (-1). Thus, as a result of a single hydrolysis step, methionine-free at position -1 G - CSF, the amino acid sequence of which at the N-terminus begins with amino acids Thr (+1) - Pro (+2) naturally occurring G - CSF.

 When G - CSF is expressed in prokaryonts, they form sparingly soluble aggregates (refractie bodies) that are inactive. Before using the protein, for example, for therapeutic purposes, it should be converted to its active form. With methods known to the person skilled in the art (cf. for example, European patent A 0219874, European patent A 0114506, WO 84/03711), the dissolution is first carried out by the addition of denaturing agents, to which the renaturation and, if necessary, further purification steps are added. The treatment of the protein of the invention with an IgA protease can be carried out before dissolution, after dissolution, or only after renaturation. If treatment with the IgA protease is to be carried out immediately after dissolution, the solvent (e.g. guanidine hydrochloride or urea) should be separated by dialysis before the IgA protease is added. However, treatment with an IgA protease is carried out mainly after renaturation, since in this case the yields of G - CSF are especially high.

 The conditions for treating G-CSF or another cleavable protein with an IgA protease alone are not critical. However, it is preferable to use G - CSF (or another protein) relative to the IgA protease in a weight ratio of 1: 1 to 100: 1. Conversion is preferably carried out in a buffered aqueous solution with a pH from 6.5 to 8.5. The concentration of the buffer solution lies mainly in the range between 50 and 300 mmol / L, if necessary, with the addition of 20-100 mmol / L sodium chloride. Mostly cleavage occurs at room temperature over 20-60 minutes.

After dissolution, renaturation and cleavage of the IgA protease, the cleavage product obtained in this way is purified mainly through an ion exchanger and large fractionation. The contamination obtained in this way and not containing methionine at position -1 G - CSF by other proteins is below 0.1%, mainly below 10 ^-3 %.

 Therefore, methionine-free at position -1 G-CSF by IgA protease cleavage can be separated or purified almost quantitatively from the methionine-containing fusion protein.

According to the method of the invention, it is possible to obtain recombinant G-CSF from prokaryonts, which is less than 0.1%, mainly less than 10 ^-3 %, is contaminated with other proteins and does not quantitatively contain G-CSF from prokaryonts, which at position -1 contains methionine.

 A subject of the invention is also a medicament based on G-CSF obtained from the prokaryonts as an active substance, if necessary together with conventional pharmaceutical carrier substances, excipients and excipients. Such a drug is particularly suitable for treatments in which the formation of granulocytes, especially neutrophils, should be stimulated.

 The pharmaceutical preparations according to the invention are mainly used as solutions for injection and infusion. This can be done as a result of the fact that a solution is already ready for injection that contains the composition of the invention. However, it is also possible to use pharmaceutical preparations in the form of lyophilisates. The latter are formed using known means suitable for injection purposes or solutions. The injection medium used is mainly water, which contains additives customary in injection solutions, such as stabilizers, dissolution agents, buffer solutions and isotonic additives, for example, physiological sodium chloride solution. Such additives are, for example, mannitol, a buffer solution of tartrate or citrate, ethanol, a complexing agent, such as ethylenediaminetetraacetic acid and its non-toxic salts, as well as high molecular weight polymers such as liquid polyethylene oxide to control viscosity. Liquid substances carriers for injection solutions should be sterile and poured mainly into ampoules.

 Finally, the present invention also includes the use of methionine-free at position -1, G-CSF from prokaryons to produce pharmaceutical preparations of the invention.

 For the case when, when splitting any fusion protein by the method according to the invention, the resulting product is obtained the protein X - Pro - A (wherein X is any amino acid and A is any amino acid sequence), which however carries an undesired X - Pro dipeptide at its amino terminus, then this undesired dipeptide can be separated as part of the method of the invention by further treatment with dipeptidyl aminopeptidases (DRAP). So far, dipeptidyl aminopeptidases have been found in a number of microorganisms, insects, amphibians and in various human tissues. They serve, for example, for the gradual chemical processing of precursor proteins and have partially pronounced specificity for the amino-terminal cleavage of the X - Pro dipeptide (X Pro - DAPARases; G. Kreil, Trendsin Biochemical Scenes 15, 23 - 26, 1990. Therefore, a combination of the method according to the invention Igases and X - Pro - DRAP can produce the desired proteins with any amino terminal acids.

 Now, using the above combination of Igase and X - Pro - DRAP, it is also possible to obtain proteins with a different N-terminal amino acid sequence from prokaryonts in position - 1 without methionine. For this, a fusion protein is first obtained that has the amino acid sequence Met - Y - Pro. ' X - Pro - A, and now in this case, the amino acid sequence A without both N-terminal amino acids X - Pro represents the desired portion of the expressed protein.

 The expression product of a prokaryon cell with the amino sequence Met - Y - Pro. '. X - Pro - A is first cleaved with an IgA protease, so that the first cleavage product with the amino sequence X - Pro - A is formed.

 Now this protein, as described above, can be treated with a dipeptidylamino peptidase, which specifically recognizes the X - Pro sequence and cleaves after Pro. Thus, a second cleavage product is formed with any sequence of amino acid A. The method according to the invention is therefore extremely useful for producing a wide variety of proteins without an N-terminal methionine residue, and is not limited only to obtaining proteins with an N-terminal X-Pro sequence, wherein X denotes predominantly Ser, Thr or Ala.

 Finally, the invention also includes recombinant DNA that contains a region encoded for the (defined) IgA protease recognition point and is suitable for incorporation into fused proteins at the transition point. In this case, we are talking mainly about chemically synthesized DNA - a fragment at the ends of which there are usually one or more suitable restrictive intersection points.

Definitions
The method according to the invention includes biotechnological production of the desired proteins. By biotechnology is meant in this regard that the desired protein or its intermediate product is obtained using genetic technology and other biotechnological methods (for example, fermentation of microorganisms).

 The desired protein is an intermediate product or end product that may find application, for example, in the field of medicine, research, environmental protection, or industrial processes or products.

 The method of the invention comprises that the desired protein is formed from a fusion protein (also referred to as a protein fusion), wherein the fusion protein or protein fusion is composed of several fusion partners covalently linked to each other. In this case, at least one of the fusion partners represents the desired protein. The sequence of fusion partners and the degree of their repetition in a fusion protein is any; however, it predominantly consists of an amino-terminal carrier protein and a carboxy-terminal desired protein.

 The carrier protein or part of the carrier serves to impart special properties to the desired protein in the form of a fusion protein. Such properties can be expressed, for example, in the increased stability of fusion proteins, which is based on structural features and, thereby, largely on higher resistance as opposed to cellular proteases, or on the transfer of fusion proteins to the environment with little proteolytic activity. In addition, the carrier protein may contain properties that provide for the efficient purification of fusion proteins. These include, for example, the binding of certain ligands in connection with the affinity chromatography method, the deposition of fusion proteins in precipitated bodies separated at low cost, and the transfer of proteins to easily accessible places.

 The areas within the fusion protein in which the components (carrier proteins and the desired proteins) of the fusion protein are connected to each other are called transition regions.

 Each transition region can be defined by one or more amino acid sequences. Amino acid sequences (like all other amino acid and protein sequences) are implied and depicted from the amino terminal (left) towards the carboxy terminal (right).

 Within the scope of the method of the invention, all those amino acid sequences in the transition regions that are to be cleaved by IgA proteases comprise a cleavage sequence or recognition sequence according to the method of the invention.

 The place between the two amino acids of the amino acid sequence in which the fusion of proteins or the fusion of proteins occurs is called the cleavage point.

 The method of the invention includes enzymatic cleavage of fusion proteins in transition regions by an IgA protease. Under the IgA protease or igase within the method of the invention is meant the IgA protease of the strain Neisseria gonorrhocae MSII and all other enzymes related to this protease at the nucleotide level and the process of its formation. IgA proteases of the genus Neisseria and Hacmophilus are also ranked here.

 The microorganism E. coli ED 8654 was deposited under the number DSM 2102 at the German Center for Microorganisms. Grisebachstrasse 8, 3400 Göttingen.

 The invention is illustrated by the following examples in combination with the drawings.

 FIG. 1 shows schematically a protein fusion between a carrier protein of MS2 polymerase (99 amino acids) and a β region (amino acid position 1195 - 1505) of an IgA protease precursor from N..gonorrhoeae MSll. The transition region between these two parts consists of 12 amino acids and contains the cleavage sequence - Pro - Pro. '. Thr - Pro - for igazy. To design the cleavage site, four oligonucleotides (1) to (4) were inserted between the restrictive intersection points of Eco RI and Hind III. The preparation of the polypeptide and cleavage with purified igase are explained in detail in Example 5.

 FIG. 2 shows a protein fusion consisting of a protein carrier of 99 amino acids of MS2 polymerase and 6 plasmid encoded amino acids, and 206 amino acids from human CD8 protein of T lymphocytes. In the amino acid sequence of the CD8 protein is a naturally occurring cleavage sequence that is cleaved with Igase (see Example 6).

 FIG. 3 shows the fusion of B63 proteins that was produced using a secretory expression system from E. coli cells. It consists of the lower part (103 amino acids) on the amino terminus of cholera toxin B, followed by the connecting region (11 amino acids) with the Igase cleavage point, and on the carboxyl terminus, part (amino acid position 1097 - 1160) of the IgA protease precursor B region. The Igase intersection point was inserted using both Tk 006 and Tk 007 oligonucleotides between both protein regions.

 FIG. 4 schematically shows the fusion of proteins B49 and B59. They consist of cholera toxin B at the bottom and B, the precursor region of IgA protease. Between these parts, they contain two different transition regions with two different igase cleavage sequences (-Pro - Pro - Ala - Pro and Pro - Pro - Thr - Pro -). The cleavage sequence of the construct with synthetic oligonucleotides (see Fig. 3).

Example 1. Construction of a plasmid for the expression of methionine-free G - CSF
The construction is carried out using an expression carrier pPZ07 mg 11 ac (WO. 86/09373). For this, the expression carrier pPZ07 - mg 11 ac is cleaved with Nco I and the protruding ends with Mung bean nuclease are removed. Then, the Bam HI carrier is cleaved. The DNA recognition sequence of IgA is obtained through the following oligonucleotides.

Олигонуклеотид B:

Обе олигонуклеотиды добавляют в эквимолярных количествах и вводят приблизительно в 100-кратном избытке в расщепленный, как описано выше, носитель pPZ07 - mg11 ac. После повторного перевязывания трансформируют обычным образом сделанные прочными клетки E. coli K12. Известными методами выделяют ДНК из клеток и расщепляют с Apal и Bam H1. Фрагмент G - CSF длиной приблизительно 520 бп выделяют при применении ограничительных эндонуклеаз Apal и Bam H1 из G - CSF - последовательности (Science 232 (1986), 61 - 65). Этот фрагмент вводят в расщепленный также с Apal и Bam H1 носитель.Oligonucleotide A:

Oligonucleotide B:

Both oligonucleotides are added in equimolar amounts and introduced in approximately a 100-fold excess into the cleaved, as described above, carrier pPZ07 - mg11 ac. After re-ligation, E. coli K12 cells that have been robust are transformed in the usual manner. Known methods isolate DNA from cells and cleave with Apal and Bam H1. A G-CSF fragment of approximately 520 bp in length was isolated using Apal and Bam H1 restrictive endonucleases from the G-CSF sequence (Science 232 (1986), 61–65). This fragment is introduced into cleaved also with Apal and Bam H1 media.

 Example 2. In addition to the IgAl recognition sequence, the fused protein may also contain peptides as an aid for purification. The latter may consist of DNA, which encodes streptavidin. For this, the streptavidin gene (WO 89/03422) is cloned into the correct translational array before the IgA protease recognition sequence.

Example 3. Using the plasmids described in Example 1, they transform E-coli K12 cells (ED 8654, DSM 2102), select on an antibiotic marker (ampicillin) and characterize the plasmids by restrictive analysis. Such a clone is used for the cultivation and expression of G - CSF. Cells are cultured in complete medium. This medium contains 1 g of 16 g of bactotryptone (Difco), 10 g of yeast extract (Difco) and 5 g of sodium chloride. Cells are allowed to grow to an OD (surface dose) of 546 2.0 and then induced with ^1-3-3 mol / L IPTG. After another 4 hours, cells were harvested by centrifugation, dissolved with lysozyme / EDTA (ethylenediaminetetraacetic acid) and G - CSF was isolated as included bodies (IB's, cf. European Patent A 0219874).

 The denaturation or renaturation of the isolated insoluble G-CSF fusion particles is carried out as described in European Patent A 0219874. Denaturation is carried out by dialysis with respect to 6 mol / L guanidine hydrochloride. Here you can select the part that is already fissile without residue and after dialysis with respect to 5 mmol / L potassium phosphate buffer solution with pH 7 can be used for cleavage with IgA protease (example 4).

 Alternatively, after denaturing in guanidine hydrochloride, dialysis is carried out against a 5 mmol / L potassium phosphate buffer solution with a pH of 7, which contains 1 mmol / L GSH and 3 mmol / L G SSG. After renaturation, dialysis relative to 5 mmol / L potassium phosphate buffer solution with a pH of 7 also occurs here.

Example 4. The cleavage of the fused protein IgAl - protease to obtain native G - CSF without methionine at position -1
The IgAl protease is isolated as described in EMBO Jour 3. (1984), 1595 - 1601. To 10 μg of the renatured or denatured according to Example 3 G - CSF, 2-5 μg of IgA protease is added and incubated for 30 minutes at room temperature. Part of various ion-exchange columns, such as Mono-Q or Mono-S, can be isolated without methionine G - CSF. The amino-terminal protein sequence indicates that purified G-CSF begins with the correct amino acid sequence Thr (+1) - Pro (+2).

Example 5. Obtaining protein fusion and cleavage of insoluble protein aggregates from the "included bodies" at a specific point of intersection of igase
The prokaryotic expression vehicle pEX31C (k. Strebel, Journal of Virology 57, 983 - 991, 1986) was modified in such a way that protein fusion super-produced with this system in E. coli cells could be cleaved with Igase into the carrier protein and the desired protein. To this end, a double-stranded DNA was prepared from synthetically prepared oligonucleotides - a fragment that encodes the amino acid sequence Thr - Pro - Ala - Pro - Arg - Pro - Pro. '. Thr - Pro. This DNA fragment was introduced using genetic technology into the intersection point of EcoR1 of the pEX31C expression carrier. In addition, two other synthetic DNA fragments were inserted directly at the border at the Hind III intersection point, which contained a number of suitable intersection points for organic endonucleases and terminal signals for bacterial enzymes involved in gene expression. Into the expression plasmids pEV37 obtained in this way, using the intersection points for SmaI and Hind III, a DNA fragment was introduced that encodes the β region of IgA, the precursor protease protein of Neisseria gonorrhoeae MSII. As a result of this, a hybrid gene was obtained, in the expression of which a fusion protein was formed in E. coli. The latter contained 99 amino acids of MS2 polymerase as a carrier protein at the amino terminus, followed by a central transition region of 12 amino acids with a igase cleavage sequence and a desired β region at the carboxyl end (see FIG. 1). Using purified igase, the β region could be cleaved from the carboxyl end of the protein fusion at the intersection point of Pro. '. Thr inside the transition region.

The poazmidus hybrid gene was introduced by transformation into E. coli cells, which, for controlled overproduction of protein fusion, contained the regulation factor Cl ⁸⁵⁷ from the bacteriophage lambda (E. Remaut, Gene 22, 103 - 113, 1983). By increasing the temperature from 28 ^° C to 42 ^° C, the Cl ⁸⁵⁷ repressor was inactivated and, as a result, the protein production in recombinant E. coli cells was activated. To do this, 50 ml of E. coli culture grown for 12 hours at 28 ^° C was transferred to 200 ml of medium preheated to 45 ^° C and cultivated for 2 hours at 42 ^° C. At this stage, protein fusion was accumulated in the bacterial cytoplasm in large amounts in the form of "included bodies". Then the bacteria were collected by centrifugation, suspended in 20 ml of lysis buffer solution (10% sucrose, 50 mmol Tris / HCl pH 8.0, 1 mmol EDTA) and after adding 400 μl of lysozyme solution (5 mg / ml) they were incubated for 30 min at 22 ^o C. Triton-X-100 detergent was added to a final concentration of 0.1% and the solution was incubated again for 30 minutes. DNA released during cell lysis was crushed by sonication, insoluble components, including protein fusion in the deposited bodies, were centrifuged and then washed in 5 ml of HINTE buffer solution (1 mol of urea, 50 mmol of NaCl, 50 mmol of Tris / HCl pH 8 , 0.1 mmol EDTA). After repeated centrifugation, the precipitate was suspended by ultrasonic irradiation in 5 ml of PBS - buffer solution (20 mmol of potassium phosphate, H 7, 5, 140 mmol of NaCl) and washed. This process was repeated several times to completely remove urea residues. Finally, the insoluble fraction that contained the fusion protein was suspended by ultrasonic irradiation in 5 ml of PBS buffer.

The amount and amount of suspended protein fusion was determined by SDS - polyacrylamide - gel electrophoresis (12.5%) and subsequent staining with Coomassie - blue. For cleavage, a suspension of proteins in the enzyme / substrate ratio 1/100 (w / w) was incubated for 3 hours at 37 ^° C. Subsequent cleavage of the crude and insoluble protein fusion was investigated analytically by SDS-polyacrylamide gel electrophoresis, and it turned out that a polypeptide of the expected size for the β-protein has formed. This protein was transferred onto a nitrocellulose membrane and subjected to automated sequence analysis. The terminal amino acid sequence confirmed its formation from protein fusion by precise cleavage at an existing igase cleavage point.

 However, only about 50% of the total amount of the introduced substrate was converted by splitting. By adding large amounts of igase and longer reaction times, no increase could be achieved. This indicates that in the non-crossed portion of the fusion protein, the intersection point for igase is not available. Hybrid protein and cleavage products and after incubation with Igase are in the form of insoluble aggregates and therefore cannot be sedimented from the suspension by centrifugation.

Cleavage yields of up to 90% were achieved when protein fusion was used instead of the contaminated “incorporated bodies” fraction, which was previously subjected to an additional purification step. For this, the insoluble precipitate after washing in H1NTE buffer solution (see above) was absorbed in 5 ml of H7NTE buffer solution (7 mol of urea, 50 mmol of NaCl, 50 mmol of Tris / HCl pH, 8, 1, 1 mmol of EDTA). Insoluble parts were separated by centrifugation, and a relatively 5 L PBS buffer solution at 4 ^° C was dialyzed into the soluble fraction. When the urea was removed by dialysis, the fused protein precipitated from the solution in the form of insoluble aggregates. Precipitated aggregates as a result of treatment with irradiation with ultrasound were transferred into a thin suspension and, as described above, were digested with Igase and analyzed.

Example 6. Specific Cleavage of Renatured, Soluble Protein Fusion with Igase
Using a pEX expression system, a hybrid protein was obtained (see example), which consists of MS2 polymerase and part of CD8, a protein of human cytotoxic T lymphocytes (see Fig. 2). After preliminary purification and dissolution of the protein from the “incorporated bodies” in the H7NTE buffer solution, the prepared 12.5% SDS polyacrylamide gel was used as the next purification step. Protein fusion as a separate band after Coomassie-blue staining was excised from the gel and then, according to the Hunkapiller method (Methodsin Enzymology 91, 227 - 235, 1983), separated from the gel material. In this case, electroelution was performed in a TAE buffer solution (40 mmol Tris / acetate, pH 7.9), to which 0.1% SDS (sodium lauryl sulfate) was added. SDS was later removed by dialysis with respect to 5 L of TAE buffer at 22 ^° C. With further dialysis, the protein was transferred to a storage buffer (20 mmol of potassium phosphate, pH 7, 5, 140 mmol of NaCl, 50% glycerol). The soluble fusion protein obtained in this way was incubated with purified igase (see Example 5) and completely digested with the cleavage point (-Pro - Pro. '. Thr - Pro - Ala - contained in the CD8 molecule, see Fig. 2) two polypeptide fragments. The specificity of the cleavage was verified by analysis of the amino acid sequence at the amino terminus of the smaller cleavage product. The result was, as expected, the sequence Thr - Pro - Ala-Pro - Thr - Ile.

 Example 7. Specific cleavage of a soluble protein obtained from supernatants of a protein fusion culture using igase.

Protein fusion consisting of choleratoxin B lower part and part of the β-region (positions 1097 - 1160; J Pohlmer, Nature 325, 458- 462, 1987) of the N. gonorrhoeae VSII IgA protease precursor was obtained in soluble form from recombinant culture supernatants E. coli cells. In order to separate both parts of the proteins from each other, an artificial cleavage sequence for igase (Pro - Pro. '. Thr - Pro-) was introduced into the transition region between the choleratoxin B - the lower part and the β-region using genetic technology methods. For this, the Tk006 and Tk007 oligonucleotides were introduced between the restriction intersection points of EcoRI and SacII in the transition region (see Fig. 3). The fusion protein was collected from 2 L of supernatant grown for 12 hours at 37 ^° C of the bacterial culture by precipitation with ammonium sulfate and then dialysis against 5 L of PBS buffer. To cleave it at a concentration of 50 μg / ml in PBS - buffer solution at an enzyme / substrate ratio of 1/50 weight / weight), incubated with purified igase was incubated for 2 hours at 37 ^° C. Immunoassay showed that more a large fragment of the cleavage in its molecular weight and in its reaction with antiserum corresponded to natural choleratoxin B - the lower part.

Example 8. Specific cleavage of protein fusions on the surface of gram-negative bacteria using igase
The secretory expression system was used to expose the fusion of the TKB 49 and TKB 59 proteins, consisting of the lower choleratoxin and the β-region IgA protease on the surface of recombinant salmonella, to the outside. The hybrid gene encoding TKB 49 contained in the transition region between the toxin region and the β region the original cleavage sequence (c) (-Pro - Pro. '. Ala - Pro -) for igase. On the contrary, artificial DNA was introduced into the gene for TkB 59 - a fragment consisting of Tk 006 and Tk 007 oligonucleotides between the restrictive intersection points of EcoRI and SacII, which encoded the cleavage sequence (-Pro - Pro. '. Thr - Pro -) (see Fig. . 3). If the intact bacteria that carried such protein fusion coupled to their surface were incubated with purified igase, then specific cleavage at the igase intersection points could be observed. In immunoassays, it was shown that small cleavage fragments appearing during cleavage in size and in reaction with antiserum corresponded to natural lower choleratoxin B.

 Example 9

 Purification of active Igase from supernatants of a culture of recombinant E. coli cells.

 Recombinant E. coli C600 cells that contain the plasmid pEX1070 (German patent 3622221.6) with a modified IgA protease gene secrete active Igase into the culture supernatant. By filtering through a membrane, the enzyme could be accumulated in the supernatant of the culture and then precipitated from the solution with ammonium sulfate (0.42 g / ml). After centrifugation, the precipitate was dissolved in a Biorex buffer solution (50 mmol of potassium phosphate, pH 7, 0, 8.6% glycerol) (1 ml of buffer solution per 1 L of culture supernatant), balanced by dialysis against 2 L of buffer solution, and then subjected to cation exchange chromatography (Biorex 70). The bound IgA protease in one step was eluted with a wash buffer (500 mmol potassium phosphate, pH 7.0, 8.6% glycerol) from the column and fractionated. The fractions containing the IgA protease were then analyzed by SDS-polyacrylamide gel electrophoresis (12.5%). At this point of purification, an average degree of purity> 90% was obtained. To obtain Igase in pure form, gel filtration was performed with Sephacryl HR300 in a Biorex buffer solution, followed by subsequent cation exchange chromatography (see above). Igase activity was checked by incubation with IgA antibodies and separation of the resulting cleavage products in SDS - polyacrylamide gel.

Первичный 1B :

Оба олигонуклеотида добавляют вместе в эквимолярных количествах и вводят приблизительно в 100-кратном избытке в расщепленный, как описано выше, носитель pPZO7-mgILac. После перевязывания сделанные обычным путем компетентными клетки E. coli K12 трансформируют. Известными методами выделяют из клеток ДНК, расщепляют при помощи SalL/BamH1 и перевязывают с ДНК - фрагментом, который содержит кодирующую область интерлейкина 3 без сигнальной последовательности (описано ниже).Example 10. Construction of a plasmid for the expression of methionine-free interleukin 3
Construction is carried out using an expression carrier pPZO7-mgLac (WO 88/09373). For this, the pPZO7-mgILac expression carrier is cleaved with NCOl and the protruding ends are removed with a Mung bean nuclease. Then, the BamHI carrier is further cleaved. The optimized amino-terminal region of the fusion protein is obtained at the DNA level through the following oligonucleotides:
Primary 1A:

Primary 1B:

Both oligonucleotides are added together in equimolar amounts and introduced in approximately a 100-fold excess into the pPZO7-mgILac cleaved, as described above. After ligation, the E. coli K12 cells made in the usual way competent are transformed. Known methods are used to isolate DNA from cells, cleave with SalL / BamH1 and bind to DNA, a fragment that contains the coding region of interleukin 3 without a signal sequence (described below).

Первичный 2B
5^,TTCGTTGGATCCCTAAAAGATCGCGAGGCTCAAAGT 3^,
Получающийся PCR-фрагмент дополнительно перерезают ферментами Sall и BamH1 и его можно вставлять непосредственно в вышеописанный носитель ДНК и связывать ковалентно при помощи лигазы.Obtaining the coding region of interleukin 3 with a recognition region for IgA protease at the DNA level is carried out using the known PCR technique, and the PCR reaction is carried out with the c-DNA of interleukin 3 as a template and with the primary described below:
Primary 2A:

Primary 2B
5 ^, TTCGTTGGATCCCTAAAAGATCGCGAGGCTCAAAGT 3 ^,
The resulting PCR fragment is further cut with Sall and BamH1 enzymes and can be inserted directly into the above-described DNA carrier and bound covalently using ligase.

 After transformation of the DNA into an appropriate host, for example E. coli K12 C600, IL 3 can be synthesized in E. coli in the form of Rb'S and then isolated. As described for G-CSF, the protein is denatured and renatured, and the renatured protein is cleaved using an IgA protease. The methionine-free IL 3 obtained in this way after further purification steps can be used for therapy.

Example 11 Construction of a plasmid for the expression of methionine-free interleukin 2
Construction can be carried out using the pPZO7 expression carrier, mgllac (WO 88/09373), which, after incorporation of primary 1A and 1B, as described in Example 10, was digested with Sall / BamH1. Obtaining the coding region of interleukin 2 with the recognition region of IgA protease at the DNA level is carried out by PCR, and PCR is carried out with the c-DNA of interleukin 2 as a template and primary 3A and 3B, which also encode the recognition region for IgA protease.

Первичный 3B :
5^,TTCGTTGGATCCTCAAGTTAGTGTTGAGATGATGCTTT 3^,
Полученный таким путем PCR - фрагмент дополнительно перерезают ферментами Sall и BamH1 и его можно непосредственно вставлять в вышеописанный носитель ДНК. Дальнейшее происходит, как описано при IL 3. В предыдущем примере было описано, как соответствующим применением точки распознавания IgA-протеазы и последующим способом можно получать не содержащие метионина терапевтические протеины, которые начинаются с последовательности кислот Ala - Pro. Другие протеины, которые аналогично вышеописанным примерам можно получать без метионина, а именно при применении олигонуклеотидов, которые содержат область распознавания для IgA-протеазы и область, которая по аналогии соответствует 5' или 3' концу опубликованной последовательности и может быть получена через PCR-увеличение. Ниже приведены другие соответствующие терапевтические протеины, которые в зрелой встречающейся в природе форме начинаются с Ala - Pro и поэтому могут быть получены аналогичным образом по способу изобретения.Primary 3A:

Primary 3B:
5 ^, TTCGTTGGATCCTCAAGTTAGTGTTGAGATGATGCTTT 3 ^,
The PCR fragment obtained in this way is further cut with Sall and BamH1 enzymes and can be directly inserted into the above-described DNA carrier. Further occurs as described in IL 3. In the previous example, it was described how, using the IgA protease recognition point and the subsequent method, methionine-free therapeutic proteins that start with the Ala-Pro acid sequence can be obtained by the following method. Other proteins, which, similarly to the examples described above, can be obtained without methionine, namely, using oligonucleotides that contain a recognition region for an IgA protease and a region that, by analogy, corresponds to the 5 'or 3' end of a published sequence and can be obtained via PCR magnification. The following are other relevant therapeutic proteins which, in a mature, naturally occurring form, start with Ala-Pro and therefore can be obtained in a similar manner by the method of the invention.

 Cathepsin L (EC 3.4.22.15), Mason, R. W. et al, Biochem. J. 240, 373 - 377, 1986.

 Erythropoietin, Lai, P.H. et al. J. Biol. Chem. 261, 3116 - 3121, 1986.

 Interleukin - 1 beta, Zsebo, K.M. et al. BLood 71, 962-968, 1988.

 Osteonectin, Fisher, L.N. et al. J. Biol. Chem 262, 9702-9708, 1987.

 Type IV Collagenase, Collier I.E. et al. J. Biol, Chem 263, 6579 - 6587, 1988.

Further, in this way it is possible to obtain proteins that in mature form begin with the sequence of amino acids Ser, Pro. An example here is
Alpha-1 Antitrypsin, Hill, RE et al., Nature 311, 175-177, 1984.

 Atrial natriuretic factor, Kambayashi, J. et al., FEBS Lett. 259, 341 - 345, 1990.

 Other examples for proteins that, in their mature form, start with Thr-Pro and can be therapeutic, for example, are, for example, Complement factor B, Campell R.D. et al., Proc. Nat. Acad Sci 80, 4464 - 4468, 1983.

 Apolipoprotein A, Eaton, D. L. et al. Proc. Nat. Acad Sci. 84, 3224 - 3228, 1987.

 The following is the deposit data for the microorganism mentioned.

Claims (19)

1. A method of obtaining a polypeptide of interest by expression and subsequent enzymatic cleavage of a fusion protein, comprising transforming a host cell with a recombinant expression vector containing a hybrid DNA fragment encoding a fusion protein with the general formula AP, where A is either methionine or any amino acid a sequence, B is any amino acid sequence, P is a transition region, including or forming, when combined with a specific protease cleavage site, not contained in the floor of the desired polypeptide, culturing transformants in a medium that expresses the recombinant product, treating it with a protease whose cleavage site is integrated into the amino acid sequence of the fusion protein, and isolating the part corresponding to the polypeptide of interest, characterized in that the cleavage site of the specific protease in the fusion protein is represented by the amino acid sequence selected from the group that includes

and for the treatment of expressed fusion protein, a JgA protease is used at a weight ratio of the enzyme to substrate of from 1: 1 to 100: 1 and a pH of from 6.5 to 8.5.  2. The method according to p. 1, characterized in that the region of the encoded fusion protein is represented by a polypeptide with high affinity for specific substances used in purification.  3. The method according to p. 1, characterized in that region A of the encoded fusion protein is part of the β-galactosidase sequence, and the host cell is E. coli. .  4. The method according to any one of paragraphs. 1-3, characterized in that for the cleavage of the fusion protein, a JgA protease from pathogenic species of bacteria of the genus Neisseria, preferably N. gonorrhoeae and N. meningitidis, is used.  5. The method according to any one of paragraphs. 1-3, characterized in that for the cleavage of the fusion protein, a JgA protease from representatives of the genus Haemophilus, preferably H. influenzae and H.aegypticus, is used.  6. The method according to any one of paragraphs. 1-3, characterized in that for the cleavage of the fusion protein, a JgA protease from a non-pathogenic recombinant producer strain is used.  7. The method according to any one of paragraphs. 4-6, characterized in that for the cleavage of the fusion protein, a JgA protease in immobilized form is used.  8. The method according to any one of paragraphs. 4-6, characterized in that they break down the fusion protein, which is both in soluble and in insoluble form.  9. The method according to p. 8, characterized in that they cleave a fusion protein in the form of insoluble inclusion bodies. 10. Hybrid DNA that encodes a fusion protein described by the General structural formula
A '- P' - B '
where A 'corresponds to the codon of methionine;
B 'corresponds to a nucleotide sequence encoding a G-CSG having the N-terminal sequence of Thr - Pro;
P 'corresponds to the nucleotide sequence encoding the transition region with the C-terminal sequence of Pro-Pro.  11. A fusion protein containing a G-CSF sequence obtained by expression in E. coli cells of the hybrid DNA of claim 10. 12. Hybrid DNA encoding a fusion protein described by the General structural formula
A '- P' - B ',
where A 'corresponds to the nucleotide sequence encoding MS2 polymerase;
B 'corresponds to the nucleotide sequence encoding a fragment (1195-1505) of the β region of the JgA protease precursor N gonorrhoeae MSII having the N-terminal sequence of Thr-Pro;
P 'corresponds to the nucleotide sequence encoding the transition region of 12 amino acids with the C-terminal sequence of Pro-Pro.  13. A fusion protein obtained by expression in E. coli cells of the hybrid DNA of claim 12. 14. Hybrid DNA encoding a fusion protein described by the General structural formula
A '- P' - B ',
where A 'corresponds to the nucleotide sequence encoding the B-subunit of cholera toxin;
B 'corresponds to the nucleotide sequence encoding a fragment (1097-1160) of the β region of the JgA protease precursor N..gonorrhoeae MSII having the N-terminal sequence of Thr-Pro;
P 'corresponds to the nucleotide sequence encoding the transition region of 11 amino acids with the C-terminal sequence of Pro-Pro.  15. A fusion protein obtained by expression in E. coli cells of the hybrid DNA of claim 14. 16. Hybrid DNA encoding a fusion protein described by the General structural formula
A '- P' - B ',
where A 'corresponds to the nucleotide sequence encoding the B-subunit of cholera toxin;
B 'corresponds to the nucleotide sequence encoding a fragment (1097-1505) of the β region of the JgA protease precursor N..gonorrhoeae MSII having the N-terminal sequence of Thr-Pro;
P 'corresponds to the nucleotide sequence encoding the transition region with the C-terminal sequence of Pro-Pro.  17. A fusion protein obtained by expression of E. coli hybrid DNA according to claim 16. 18. Hybrid DNA encoding a fusion protein described by the General structural formula
A '- P' - B ',
where A 'corresponds to the nucleotide sequence encoding the B-subunit of cholera toxin;
B 'corresponds to the nucleotide sequence encoding a fragment (1097-1505) of the β region of the JgA protease precursor N..gonorrhoeae MSII having the N-terminal sequence of Ala-Pro;
P 'corresponds to the nucleotide sequence encoding the transition region with the C-terminal sequence of Pro-Pro.  19. A fusion protein obtained by expression in E. coli cells of a hybrid DNA according to claim 18. Priority on points:
05.17.90 according to claims 1-9, 12-19;
12/10/90 according to 10 and 11.

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