JPH08187094A - Production of protein - Google Patents

Abstract

PURPOSE: To efficiently obtain the objective protein by transforming a host cell with an expression vector containing a gene of a fused protein comprising a protective polypeptide and an object polypeptide, manifesting the gene and cutting with an endogenous protease of the host. CONSTITUTION: A host cell (e.g.; Escherichia coli) is transformed by an expression vector integrated a gene coding fused protein expressed by the formula: A-L-B (A is a protective polypeptide; B is the object polypeptide; L is a linker peptide having a substrate specifically recognized by an endogenous protease of a host cell) or the formula: A-L-B-L-C (C is a protective peptide) and comprising a protective polypeptide and the object polypeptide, cultured and a gene is manifested, the cell is crushed and the fused protein is separated, then the fused protein is solubilized and the linker peptide is cut with the endogenous protease of the host cell (e.g.; Escherichia coli ompT protease), thus the object polypeptide is efficiently obtained from the fused protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a target polypeptide using a gene recombination technique. Preferably, the polypeptide of interest is a bioactive polypeptide, for example an enzyme such as a protease. As an example, the present invention relates to a method for producing a polypeptide of interest, which is a Staphylococcus aureus V8 protease derivative.

[0002] As a more specific example, Staphylococcus aureus ( Staphylococcus aureus) using the E. coli expression system
coccus aureus ) V8 protease-derived V8 protease derivative is expressed as an insoluble fusion protein, and the V8 protease derivative is cleaved from the fusion protein by an endogenous ompT protease derived from Escherichia coli in the presence of a denaturing agent. It is a method for producing an active V8 protease derivative through refolding.

[0003]

[ Prior Art] Staphylococcus aureus ( Stap
hylococcus aureus) (hereinafter S. aureus (S. aure
us )) V8 protease is a type of serine protease secreted by the S. aureus V8 strain into the medium. This enzyme
In 1972, it was isolated and purified by Drapeau, GR and others as a kind of serine protease that specifically cleaves the C-terminal side of glutamic acid and aspartic acid secreted in the medium of S. aureus V8 strain (Jean Houmard and Gabriel.
R. Drapeu (1972) Proc. Natl. Acad. Sci. USA 69, 35
06-3509), in 1987 Cynthia Carmona et al. Revealed the DNA base sequence of this enzyme (Cynthia
Carmona and Gregory L. Gray (1987) Nucleic Acids
Res. 15, 6757).

It is considered that this enzyme is expressed as a precursor consisting of 336 amino acid residues, and then 68 prepro sequences are removed from the N-terminal and secreted as a mature protein. Further, it is known that this enzyme has a proline-aspartic acid-asparagine repeat sequence in its C-terminal region (amino acid numbers 221 to 256). It is unclear whether this repetitive sequence is necessary for enzyme activity, and it is thought that Gray et al. May function when it exists as an inactive enzyme before it is secreted. There is.

[0005] Despite the insufficient functional analysis as an enzyme, this enzyme specifically cleaves the peptide bond at the C-terminal side of glutamic acid and aspartic acid, and thus is widely used for determining the amino acid sequence of proteins. ing. In addition, since this enzyme acts on the substrate even in the presence of urea (about 2M), the fusion protein expressed in a large amount as an insoluble fusion protein by the gene recombination technology is solubilized with urea, and then the enzyme acts It is also used in the step of releasing the desired peptide from the fusion protein.

The present inventors have succeeded in efficiently producing human calcitonin by using the above-described method for producing human calcitonin by the gene recombination method (JP-A-5-328).
992). Also, in an example in which human glucagon was expressed as a fusion protein in an Escherichia coli expression system, S. aureus V8 protease was used to excise human glucagon from the fusion protein (Kazumasa Yosikawa et al. (1992) Journa
l of Protein Chemistry, 11, 517-525).

[0007] As described above, although the present enzyme has been used in a great number of studies and in the production of peptides by gene recombination, it has been purified from the culture solution of S. aureus V8 strain. There are problems that other proteases are mixed in a trace amount, (2) purification is required from a pathogenic bacterium, and (3) it is expensive.

[0008]

Accordingly, the present invention is directed to the large scale production of a polypeptide of interest, such as S. aureus V8 protease. It is very useful in research and industry to produce a highly purified target polypeptide such as S. aureus V8 protease, and a large-scale industrial production of the polypeptide is highly desired.

[0009]

In order to achieve the object of the present invention, a gene encoding a target polypeptide, for example, a gene for S. aureus V8 protease from S. aureus V8 strain is isolated and a gene recombination technique is used. It is necessary to produce and purify the polypeptide, for example, the enzyme in a large amount at low cost by using a safe host cell such as Escherichia coli.

Generally, in order to produce a desired polypeptide or protein by a genetic engineering method, the polypeptide produced by the host adversely affects the survival or proliferation of the host and further the expression of the desired polypeptide or protein. In order to prevent this, and to facilitate isolation and purification of the expressed target polypeptide or protein, the target polypeptide or protein is produced as a fusion protein, which is used as an insoluble inclusion body in the host cell. It may be preferable to accumulate the

However, the fusion protein containing the desired polypeptide or protein does not always form an inclusion body. Therefore, in one embodiment of the present invention, the desired polypeptide or protein is expressed as a fusion protein with another protein and accumulated as an inclusion body in the host cell. Although the protease originally possessed by the host cell cannot act pairwise on the inclusion body, after isolation and lysis of the inclusion body, the host-derived protease associated with the inclusion body acts on the fusion protein. Can be cleaved to release the desired polypeptide or protein. Thus, according to the present invention, the target polypeptide or protein can be efficiently produced.

However, when the desired polypeptide or protein is made into a fusion protein by a conventional method, the fusion protein does not always form an inclusion body. In such a case, in the present invention, inclusion bodies are formed by forming a fusion protein by linking protective peptides to the N-terminal side and the C-terminal side of the target polypeptide or protein. Therefore, in the present invention, a method for efficiently producing a large amount of S. aureus V8 protease in an Escherichia coli expression system by using a genetic engineering method will be described as a specific example.

First, the present inventors considered that the expression method by the fusion protein method having no enzymatic activity is suitable for producing a large amount of S. aureus V8 protease by using the Escherichia coli expression system. Because this enzyme is a proteolytic enzyme, it is considered that if this enzyme is directly expressed in the cells, the E. coli protein is decomposed, the growth of the bacteria is stopped, and a large amount of S. aureus V8 protease cannot be obtained. Is. Therefore, it was expressed as a fusion protein having no enzymatic activity, and then the S. aureus V8 protease part was cleaved from the fusion protein using another protease, and refolding was carried out if necessary for the expression of the activity. We considered purifying V8 protease.

Further, as another protease used for excision from this fusion protein, the present inventors considered the use of ompT protease, which is considered to exist in the outer membrane of Escherichia coli. This is because adding another enzyme newly to produce S. aureus V8 protease at low cost is considered to be very disadvantageous in terms of cost. Production by E. coli using ompT protease is (1) simple manufacturing process, (2) it is not necessary to add another protease to the reaction system, and the cost is low. (3) E. coli expression system When cleaving various fusion proteins produced with V8 protease produced using Escherichia coli as a host, it is conceivable that V8 protease-producing host-derived protein (S. aureus-derived protein in commercially available V8 protease) may be incorporated into V8 protease. Needless to say, it is very advantageous in terms of not having to do so.

Escherichia coli ompT protease is a protease that exists in the Escherichia coli outer membrane fraction and selectively cleaves between basic amino acid pairs (Keijiro Sugimura and Tatsuro Nishi).
hara (1988) J. Bacteriol. 170, 5625-5632). Sugimur
For a, etc., ompT protease was purified, and various peptides were cleaved with ompT protease using 50 mM phosphate buffer (pH 6.0) at 25 ° C. for 30 minutes, and arginine-arginine, lysine-lysine, arginine-lysine and It is reported that the enzyme cleaves between the basic amino acid pair of lysine-arginine.

However, it is not mentioned whether ompT protease has activity in the presence of urea (urea concentration of 2 M or more). Since ompT protease is present in the outer membrane, after producing S. aureus V8 protease as an inclusion body in Escherichia coli, the insoluble fraction containing the inclusion body was separated by centrifugation after crushing the cells. Protease is also considered to co-precipitate.

Urea is added to the thus obtained precipitate fraction to solubilize the fusion protein of S. aureus V8 protease. If the activity of ompT protease can be retained under these conditions, the ompT protease can be used for excision of S. aureus V8 protease from the fusion protein, and further excised S. aureus V8 protease can be used as the active form. S for refolding to convert.
It was thought that aureus V8 protease could be produced in large quantities by a simple process.

The present invention will be described below. S. aureus V 8
From the gene sequence of the protease gene, it has been reported that there is a signal sequence (pre-sequence) necessary for secretion and a pro sequence of unknown function at the N-terminal side of the mature protein, and the above-mentioned repeat sequence at the C-terminal. There is. Therefore, the gene I from the N-terminal to the C-terminal of the mature protein and the gene II lacking the repeat sequence of unknown function at the C-terminal side of the mature protein were prepared. It is not known whether this repetitive sequence of unknown function is required for enzyme activity, but if it is not necessary, lowering the molecular weight of the fusion protein can increase the number of expressed proteins per cell and increase the amount of expressed protein. I thought it would be possible.

Therefore, Staphylococcus aureus V8 strain (AT
CC27733) to isolate chromosomal DNA and perform PCR 2
Two kinds of V8 protease derivative genes I and II were prepared. In order to express these derivatives, expression plasmids pV8RPT (+) and pV8RPT (-) using E. coli β-galactosidase derivative as a protective peptide of the fusion protein were prepared. In these expression plasmids, as a linker peptide gene between the Escherichia coli β-galactosidase derivative protein gene and the V8 protease derivative gene (I or II), a linker gene containing an arginine-arginine residue which is a recognition and cleavage amino acid sequence of ompT protease (for example, The fusion protein into which the R6 linker gene) has been inserted is expressed under the control of the lactose promoter.

In the fusion protein designed in this way,
After the V8 protease derivative was expressed as an insoluble fusion protein, the fusion protein was solubilized with urea, and the ompT protease cleaved the fusion protein when the urea concentration was lowered, resulting in Escherichia coli β-galactosidase derivative protein and V8 protease derivative protein. Because it was thought that they could be separated. The expression plasmid designed as described above was prepared, and expression was induced using IPTG (isopropyl-β-D-thiogalactopyranoside; hereinafter IPTG) using Escherichia coli JM101 as a host. Both of them did not become insoluble, and after crushing the bacterial cells, enzyme activity was observed in the supernatant fraction.

On the other hand, the proliferation of the bacterial cells was remarkably reduced after the expression was induced. Analysis by SDS polyacrylamide gel electrophoresis revealed that intracellular proteins were degraded by the enzymatic activity of the expressed V8 protease derivative. Therefore, this fusion protein expression method of E. coli β-galactosidase derivative and V8 protease derivative is
It was revealed that (1) the expressed protein has an enzymatic activity and causes growth inhibition of the host, and (2) the expression level of the fusion protein is very low, which is not suitable as a method for producing a V8 protease derivative. .

However, these results indicate that (1) the N-terminal pro sequence may not be involved in the folding of V8 protease, and (2) that the C-terminal repeat sequence is not involved. Since the V8 protease derivative II is also active, it was considered that this repeat sequence may not be necessary for the V8 protease enzyme activity. Next, the present inventors express V8 protease as an insoluble fusion protein in an Escherichia coli expression system, solubilize it with urea, and then use a protease to release the V8 protease portion from the fusion protein in the presence of urea. In order to fold and produce V8 protease having enzymatic activity, the following working hypothesis was set and the experiment was started.

(1) When the above-mentioned Escherichia coli β-galactosidase derivative is fused to the N-terminal side of V8 protease, it does not become an insoluble fusion protein. Therefore, in such a case, it is considered to further add a protective peptide in order to form an insoluble fusion protein, and the kanamycin resistance gene (Nucleic Acids Res. (1988) 16, 358-358) which is transposon 903 as the protective peptide is considered. 3) -derived aminoglycoside 3'-phosphotransferase protein. That is, by adding a part of aminoglycoside 3′-phosphotransferase protein to the C-terminal of a fusion protein consisting of Escherichia coli β-galactosidase derivative and V8 protease via a linker peptide, insolubilization of the fusion protein is promoted to form inclusion bodies. I thought to do it.

(2) V from the produced fusion protein
In order to enzymatically release the 8 protease moiety, the R
It was thought that the V8 protease could be dissociated from the fusion protein by arranging 6 linkers on the N- and C-terminal sides of V8 protease and cleaving with ompT, which cleaves between a pair of basic amino acids.

Therefore, based on the above working hypothesis, the construction of a novel expression plasmid was started, and a fusion protein prepared by fusing a part of aminoglycoside 3'-phosphotransferase with an Escherichia coli β-galactosidase derivative-V8 protease derivative was prepared. The expressing plasmid pV8D was created.
The V8 protease derivative encoded by this plasmid (hereinafter referred to as V8D protein) was obtained by deleting 8 amino acids from the C terminus of the above V8 protease derivative II. Is fused to a portion of the E. coli β-galactosidase derivative and aminoglycoside 3′-phosphotransferase via the R6 linker peptide.

E. coli JM101 harboring pV8D was cultured to obtain IPTG
It was revealed from the analysis by SDS PAGE that the 60-kilodalton fusion protein that was expressed when the expression was induced by E. coli formed insoluble inclusion bodies in the cells. Therefore, it was revealed that fusion of a part of the aminoglycoside 3′-phosphotransferase protein to the C-terminal side of the V8D protein causes insoluble inclusion body formation. Next, after crushing the cells, centrifugation is performed to separate the inclusion bodies, and the fusion protein is solubilized using a denaturing agent. As the denaturing agent, urea, guanidine hydrochloride, a surfactant or the like can be used.

In the examples of the present application, inclusion bodies were solubilized with 8 M urea, diluted to 4 M urea concentration, and incubated at 37 ° C. for 2 hours. The E. coli endogenous ompT protease cleaves the fusion protein under these conditions, causing β-
It was revealed by SDS PAGE analysis that bands of 12 KDa, 26 KDa and 22 KDa corresponding to the galactosidase derivative, the V8D protein and a part of the aminoglycoside 3'-phosphotransferase protein were produced, respectively.

On the other hand, W3110M which is an ompT-deficient mutant strain
As a result of a similar experiment using 25 as a host bacterium, the above band could not be detected, and it was revealed that the Escherichia coli endogenous ompT cleaves the fusion protein specifically. Furthermore, in order to confirm that it was specifically cleaved by ompT, 26 KD corresponding to V8D protein was confirmed by SDS PAGE.
The band a was cut out from the SDS PAGE gel and the N-terminal amino acid sequence was determined. It was confirmed that cleavage occurred between the arginine and arginine sequences of the R6 linker peptide.

Therefore, the fusion protein and E. coli endogenous ompT
Protease is present in the inclusion body fraction of the precipitate generated by centrifugation, and has sufficient enzymatic activity even in the presence of 4M urea after solubilization with a denaturing agent, especially with 8M urea. It was revealed for the first time by the present invention that the exactly expected amino acid sequence can be cleaved. Since the V8 protease derivative protein produced in the presence of urea is in a denatured state, it is considered that the enzyme activity is very low. Therefore, it was examined whether V8D protein having enzymatic activity could be obtained by refolding the V8D protein by lowering the concentration of the denaturant if necessary for expression of activity.

The sample after the ompT cleavage reaction was diluted 20-fold with 0.4M potassium phosphate buffer (pH 7.5), and then left overnight on ice. By this operation, V8D protein is about 20
% Refolding and recovery of enzyme activity was seen.
When the sample after this operation was analyzed by SDS-PAGE, the V8D protein was mainly present after refolding. This is because β-galactosidase derivative, a part of aminoglycoside 3′-phosphotransferase protein and Escherichia coli-derived protein are present in the sample before refolding, but after refolding, V8D protein having protease activity is mixed with other proteins. It is thought that this is due to the decomposition of. This result is very advantageous in purifying V8D protein after refolding.

Next, V which became the active type by the above-mentioned operation
It was examined whether the 8D protein has the same substrate recognition as the native S. aureus V8 protease. Substrate (eg,
Human calcitonin precursor fusion protein) was refolded V8D protein and native enzyme at 30 ° C.
After reacting for 1 hour, the peptide fragment generated from the fusion protein cleaved by the enzyme was analyzed by high performance liquid chromatography. As a result, it was revealed that the peptide fragments generated from both enzymes showed the same elution pattern, and that the V8D protein produced by the above method had the same substrate specificity as the natural type.

In order to express S. aureus V8 protease as an insoluble inclusion body in the cells, it is necessary for the fusion protein to have no enzymatic activity as a proteolytic enzyme. On the other hand, after refolding, Must have enzymatic activity. This seemingly contradictory property is required for the V8 protease derivative protein. For the V8D protein, a fusion protein was prepared using the N-terminal to the 212th amino acid of the native V8 protease, and the V8 protease portion was further extended to the C-terminal side to form a fusion protein with a part of the aminoglycoside 3′-phosphotransferase protein. It was examined whether or not reactivation by inclusion body formation and refolding occurs in the case of producing.

The present inventors have developed a plasmid (pV8H, pV8F, pV8A, pV8) expressing a fusion protein in which the C-terminal side of the above V8 protease derivative is extended by 2, 4, 6 and 8 amino acids.
D2, and pV8Q) were prepared by PCR and gene cloning. E. coli strain JM101 was transformed with these plasmids to prepare recombinants. The obtained strain is cultivated and IP
When expression was induced using TG, only the strains harboring the above-mentioned pV8D, pV8H and pV8F plasmids formed the inclusion body of the fusion protein, and the enzyme reactivates in refolding after cleavage of the fusion protein by ompT. It was made possible.
Inclusion body formation did not occur in the other strains, and V8 protease activity was observed in the soluble fraction after disruption of the cells.

Therefore, in order to express the V8 protease derivative protein by the fusion protein expression method to form an inclusion body and then enzymatically cleave it and perform refolding, S. aur is used.
It became clear that fusion was required before phenylalanine, which is the 215th amino acid from the N-terminal of the eusV8 protease protein. The present invention has been specifically described above by taking V8 protease as an example of a target polypeptide or protein. However, by applying the same principle and method to other target polypeptides or proteins, the desired target polypeptide can be obtained. Alternatively, the protein can be efficiently produced.

Therefore, according to the present invention, a host cell is transformed with an expression vector having a gene encoding a fusion protein consisting of a protected polypeptide and a target polypeptide, the gene is expressed, and an endogenous protease of the host cell is used. It relates to a method for producing a target polypeptide, which is obtained by cleaving the target polypeptide from the fusion protein.

Furthermore, the present invention provides a fusion protein of the formula (1) A-
LB or the formula (2) A-L-B-L-C (wherein A and C are protected polypeptides, B is a target polypeptide, and L is a substrate specifically recognized by a host cell endogenous protease. The present invention relates to a method for producing a target polypeptide B from the fusion protein by cleaving the fusion protein at the region of the linker peptide L. Here, the target polypeptide is, in addition to the aforementioned S. aureus V8 protease,
For example, physiologically active peptides such as motilin, glucagon, adrenocortical hormone, adrenocorticotropic hormone releasing hormone, secretin, growth hormone, insulin, growth hormone secreting hormone, pazopressin, oxytocin, gastrin, glucagon-like peptide (GLP-
1), glucagon-like peptide (GLP-2), glucagon-like peptide (7-36 amide), cholecystokinin,
Vasoactive Intestinal Polypeptide (va
soactive intestinal polypeptide; VIP),

Pituitary adenolatecyclase activating polypeptide
polypeptide), gastrin releasing hormone, galanin,
Thyroid-stimulating hormone, luteinizing hormone-releasing hormone,
Calcitonin, parathyroid hormone (PTH, PTH (1
-34) PTH (1-84)), peptide histidine isoleucine; PH
I), neuropeptide Y, peptide YY, pancreatic polypeptide, somatostatin, TG
F-α, TGF-β, nerve growth factor, fibroblast growth factor, relaxin, prolactin, atrial natriuretic peptide (ANP), B
-B-type natriuretic peptide (BNP)
), C-type diuretic peptide (C-type natriuretic peptid
e; CNP), angiotensin, brain-derived neurotrophic factor (BDNF), and further examples include enzymes such as KEX2 endoprotease.

In a preferred embodiment of the present invention, when the target polypeptide is a physiologically active polypeptide, preferably the physiologically active polypeptide is an enzyme, and more preferably the enzyme is a proteolytic enzyme. Is mentioned. In a most preferred embodiment as a specific example, the target polypeptide is a proteolytic enzyme, and the target polypeptide is expressed in a host cell as an inactive fusion protein, the cell is crushed, and the fusion protein is After separation, the fusion protein is solubilized with a denaturing agent that solubilizes the fusion protein, and then the target peptide is obtained from the fusion protein by cleaving the linker peptide region with host cell endogenous protease. There is a method.

In a further preferred embodiment, when the target polypeptide is a proteolytic enzyme, the target polypeptide is expressed as an inactive fusion protein in a host cell, and the cell is crushed to separate the fusion protein. Then, the fusion protein is solubilized with a denaturing agent that solubilizes the fusion protein, and then the concentration of the denaturing agent is reduced to such an extent that the endogenous protease activity derived from the host cell can be expressed, and the linker peptide L is added. A method for producing the desired polypeptide from the fusion protein by cleaving with the endogenous protease can be mentioned. In any of the above cases, it is preferable that the endogenous protease derived from the host cell be present in the same fraction as the fusion protein in the separation operation after disruption of the cells.

The protected polypeptide is not particularly limited as long as it can be expressed as a fusion protein with the protected polypeptide when the desired polypeptide is expressed. For example, Escherichia coli β-galactosidase-derived polypeptide or transposon 903 is used. Examples thereof include aminoglycoside 3′-phosphotransferase-derived polypeptides, and if necessary, those polypeptides can be used in combination as a protected polypeptide.

The linker peptide may be any linker peptide having a site that is specifically recognized by the endogenous protease of the host cell having the expression vector of the desired polypeptide. As a preferred embodiment of the linker peptide, for example, the linker peptide contains a basic amino acid pair sequence having one basic amino acid residue or consecutive two basic amino acid residues, and the linker peptide has 2 to 50 amino acids. It is preferably a polypeptide consisting of residues, and may include the basic amino acid pair at the N-terminal portion and / or the C-terminal portion of the linker peptide.

The denaturing agent for solubilizing the fusion protein also need not be specified in particular, and examples thereof include urea, guanidine hydrochloride or a surfactant. It is preferable to use urea, and the urea concentration in this case is preferably 1-8M. Further, the urea concentration after solubilizing the fusion protein is not particularly specified as long as the endogenous protease activity derived from the host cell can be expressed.

The present invention also provides a method for producing a target polypeptide, which is characterized in that the target polypeptide is cleaved from the fusion protein using the host cell-derived endogenous protease. The target polypeptide does not have to be specified. That is, the protease may be used as long as it can be used for processing the target polypeptide or protein from the fusion protein after expressing various target polypeptides or proteins as insoluble fusion proteins, and examples thereof include proteolytic enzymes. And preferably Escherichia coli ompT protease and the like as shown in the following examples.
The target polypeptide is preferably a polypeptide consisting of 20 to 800 amino acid residues, and examples thereof include S. aureus V8 protease and / or the derivative thereof as shown in the following Examples.

As a result of the above, it was revealed that the V8 protease derivative protein can be produced particularly in the Escherichia coli expression system in the method for producing a target polypeptide using the gene recombination technique according to the present invention. That is, as a preferred embodiment of the method for producing the target polypeptide, the following production steps can be mentioned. Ie;

(1) The fusion protein comprises a protected polypeptide, a target polypeptide and a linker peptide, and the protected polypeptide is derived from Escherichia coli-derived β-galactosidase-derived polypeptide and / or transposon 903-derived aminoglycoside 3′-phosphotransferase. The polypeptide of interest is S. aureus V
8 is a protease derivative and encodes the fusion protein, wherein the linker peptide region between the protected polypeptide and the target polypeptide is a linker peptide region having a substrate specifically recognized by a host cell endogenous protease. Escherichia coli which is a host cell is transformed with an expression vector having a gene,

(2) The fusion protein is expressed in Escherichia coli with the S. aureus V8 protease derivative in an inactive state, (3) the cells are crushed, and the fusion protein is separated.
A fraction containing the E. coli ompT protease, which is an intracellular protease, and the fusion protein is present, (4) solubilizing the fusion protein with a denaturing agent,

(5) After solubilization, the concentration of the denaturant is lowered to a state where the activity of Escherichia coli ompT protease is expressed, and the linker peptide region is cleaved with the protease to obtain the desired polypeptide from the fusion protein, And a step of causing refolding so that the target polypeptide becomes an active form, if necessary. A manufacturing method can be mentioned.

As a method for purifying this protein after the refolding of the V8 protease derivative, it can be purified to a high purity by a usual protein purification operation such as gel filtration, ion chromatography, hydrophobic chromatography and the like. Furthermore, it goes without saying that the V8 protease derivative shown in the examples is extremely easy to purify because the derivative is the main protein component of the reaction component at the end of the refolding reaction.

[0049]

The present invention will be described in detail below with reference to Examples. Example 1. Isolation of S. aureus V8 protease gene To isolate the V8 protease gene by PCR,
The gene was isolated by the PCR method based on the reported DNA nucleotide sequence. Three types of PCR primers having the sequences shown in Fig. 1 (b) were designed and synthesized by a DNA synthesizer (Applied Biosystems 392 type). Primer
I, II and III correspond to the V8 protease gene region shown in FIG. 1 (a), and Xho I or Sal I restriction enzyme sites are provided on the 5'side.

Jayaswal, RK et al. (J. Bacteriol. 172:
5783-5788 (1990)) isolated and prepared Staphylococcus aureus V8
PCR was performed using the chromosome of the strain (ATCC27733) and these PCR primers. 1.0 μM primer, 1 μg chromosomal DNA, 50 mM KCl, 10 mM Tris-HCl, pH8.3, 1.5 mM MgC
l2, 0.01% gelatin, 200 μM dNTP (dATP, dGTP, dC
A mixture of TP and dTTP) was added to 50 μl of the reaction mixture, and 2.5 units of Taq DNA polymerase was added to the mixture.
PCR was carried out for 30 cycles of 2 minutes at 55 ° C and 2 minutes at 55 ° C.

As a result, primer I and primer II were used to prepare a mature body containing no repeat sequence but prepro sequence.
V8 protease gene (protease gene derivative I,
0.8 kb) was obtained, and the V8 protease gene lacking the prepro sequence and the repeat sequence was obtained by primer I and primer III (V8 protease gene derivative II, 0.7 kb).
was gotten. Next, these genes were subjected to agar gel electrophoresis, purified using SUPREP-2 (Takara Shuzo Co., Ltd.), and then digested with restriction enzymes XhoI and SalI to produce V8 protease derivative genes having sticky ends XhoI and SalI. Fragments I and II were prepared.

Example 2. Expression vector pV8RPT (+) and
And pV8RPT (-) and expression of V8 protease derivative pG97S4DhCT [G] R6 used in this example is an Escherichia coli β-galactosidase derivative and human calcitonin precursor (hCT [G]).
And a plasmid pBR322 and a plasmid pG9 that highly expresses a fusion protein with
It can be prepared from 7S4DhCT [G] (see JP-A-5-328992 and FIG. 2). Plasmid pG
Escherichia coli W3110 strain containing 97S4DhCT [G] has been deposited as Escherichai coli SBM323 on August 8, 1991 under the Budapest Treaty under the Institute of Biotechnology, Agency of Industrial Science and Technology, and the deposit number is Micromachine Research Institute Article 3503.
No. (FERM BP-3503) is given.

In order to express the V8 protease gene derivatives I and II obtained by PCR, pG97S4DhCT [G] R
6 was treated with XhoI and SalI, and a DNA fragment (3.1 kb) lacking the human calcitonin precursor gene part was prepared by agar gel electrophoresis. This DNA fragment and the previously obtained XhoI and Sa
The V8 protease gene fragment with the sticky end of lI was added to T4 DNA.
Ligated with ligase, transformed into JM101, and cloned V8 protease gene derivative I pV8RPT
(+) And pV8RPT (-) in which V8 protease gene derivative II was cloned were prepared (Fig. 3).

The host strain of the plasmid is JM101 strain (this strain is, for example, Takara Shuzo Co., Ltd., In Vitrogen Catalog N
o.c660-00 etc.) was used. The amino acid sequence of the fusion protein of the V8 protease derivative and the β-galactosidase derivative expressed from these plasmids is shown in FIG. After culturing JM101 / pV8RPT (+) and JM101 / pV8RPT (-) in 100 ml of LB medium (0.5% yeast extract, 1.0% tryptone, 0.5% NaCl) at 37 ° C until OD660 reached 1.0, isopropylthiogalacto was prepared. Pyranoside (IPTG) was added at a final concentration of 2 mM to induce expression. After culturing for 2 hours after the addition, the bacterial cells were collected by centrifugation and TE was adjusted to OD660 of 5.
The cells were suspended in a buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA).

This suspension was crushed by an ultrasonic crusher (Cellruptor; Tosho Electric Co., Ltd.), and the insoluble fraction was removed by centrifugation at 12000 rpm for 5 minutes, and the supernatant fraction was used as a crude enzyme solution. used. Synthetic substrates (Z-Ph
e-Leu-Glu-4-nitranilide (Boehringer Mannheim) was used. Mix 940 μl of 100 mM Tris-HCl (pH8.0) buffer with 20 μl of 10 mM Z-Phe-Leu-Glu-4-nitranilide solution (DMSO solution), add 40 μl of crude enzyme solution, and room temperature for 5 minutes. The increase in absorption at 405 nm due to the reaction was measured. A Hitachi spectrophotometer U-3200 was used for the measurement.

As a result, JM101 / pV8RPT (+) and JM101 /
With both pV8RPT (-), 8 μg / ml of enzyme activity was observed in the crude enzyme solution prepared from those cells, and it was active in the form of a fusion protein with β-galactosidase derivative and lacking the prepro sequence. Turned out to be. It was also found that pV8RPT (-) lacking the C-terminal repeat sequence showed activity, and therefore this sequence is not essential for activity.

JM101 / pV8RPT (-) and JM101 / pV8RPT (+)
The amount of V8 protease derivatives I and II produced by E. coli is low, and SDS-polyacrylamide gel electrophoresis (SDS-
The purification operation is difficult because the band cannot be confirmed by PAGE). Considering that bacterial growth is stopped with the addition of IPTG and that the high-molecular-weight protein is decreased in the induced bacteria compared to the non-induced bacteria, the intracellularly expressed V8 protease derivative It is considered that the cause of the low expression level is lethal.

Example 3. Construction of expression vector pV8D Since V8 protease has activity even when the β-galactosidase derivative is fused to the N-terminal side as described above, it can be inactivated by fusing only the N-terminal side. Absent. Therefore, a part of aminoglucoside 3′-phosphotransferase was further fused to the C-terminal side.
An attempt was made to inactivate the V8 protease. The EcoRV site before the repeat sequence was used for fusion on the C-terminal side.

Plasmid relating to V8 protease derivative
pV8D was prepared by the procedure shown in FIG. Bgl from pV8RPT (-)
II-Sal I fragment (3.0 kb) and EcoRV-Bgl II fragment (0.7 k
b) was prepared and Nar I-Sa prepared from pG97S4DhCT [G] R10 was prepared.
By ligating with the I fragment (0.2 kb), pV8hCT [G]
I got In addition, pG97S4DhCT [G] R10 is the above pG97S4DhCT
Similar to [G] R6, plasmid pBR322 and plasmid p
It can be prepared from G97S4DhCT [G] (see JP-A-5-328992 and FIG. 2).

Next, the hCT [G] portion (0.1 kb BstE II-Sal I fragment) of the obtained pV8hCT [G] was inserted into pUC4K (Vieira, J. an.
d Messing, J., Gene 19, 259 (1982), Pharmacia Ca.
No. 27-4958-01) was replaced with the 0.8 kb SmaI-SalI fragment containing the aminoglucoside 3′-phosphotransferase gene region to prepare pV8D. Expressed from this plasmid
The amino acid sequence of the V8 protease derivative fusion protein (V8D fusion protein) is shown in FIG.

The present fusion protein has a structure in which a β-galactosidase derivative and a part of aminoglucoside 3′-phosphotransferase are fused to the N-terminal and C-terminal of V8 protease via an R6 linker sequence, respectively. The R6 linker has the following sequence: RLYRRHHRWGRSGSPL
It has RAHE (SEQ ID NO: 1) and has a structure in which the central peptide bond of RR in the sequence is cleaved by E. coli OmpT protease.

Example 4. Expression of V8D fusion protein E. coli JM101 transformed with pV8D by a conventional method
(JM101 / pV8D) in 100 ml of LB medium at 37 ° C with OD660 of 0.
After culturing up to 6, IPTG was added to a final concentration of 2 mM to induce the production of V8D fusion protein. After the addition, the culture was continued for another 2 hours, and then the cells were collected by centrifugation. For this strain, JM101 / pV8RPT (+) and JM101 / pV8R
Unlike PT (-), there was no phenomenon in which bacterial cell growth stopped with the induction of fusion protein expression, and no V8 protease activity was observed in the bacterial cells.

It was observed by a microscope that inclusion bodies were formed in the cells. 16 of the insoluble and soluble fractions obtained by ultrasonic disruption of cells before and after induction
The results of% SDS-PAGE are shown in FIG. 60kD for cells after induction
It can be seen that the V8D fusion protein of a is highly expressed and that it is present in the insoluble fraction because it forms an inclusion body.

Since a part of aminoglucoside 3'-phosphotransferase was fused to the C-terminal side of the V8 protease derivative, the cell could not take the original three-dimensional structure in the cell and became inactive, and the cell growth was not inhibited.
It was considered that high expression was achieved up to the level of forming inclusion bodies.
In addition to the 60kDa V8D fusion protein, a 27kDa protein was also found in the insoluble fraction. The protein was separated from the SDS PAGE gel and the amino acid sequence was examined.
It was found to be a degradation product containing the methionine after the 282nd position from the end.

Example 5. OmpT protection of V8D fusion protein
Processing with ase The bacterial cells obtained by the culture shown in Example 4 were suspended in 10 ml of TE buffer and then sonicated to disrupt the bacterial cells. Then, the inclusion body was recovered by centrifugation. The obtained inclusion body was suspended again in 10 ml of deionized water and then centrifuged to wash the inclusion body. The inclusion body was diluted with deionized water so that the OD660 value was 100, and 150 μl was collected and
25 μl Tris-HCl (pH 8.0), 1M dithiothreitol (DTT
) 2.5 μl and 120 mg urea to dissolve the inclusion bodies, and then add deionized water to 500 μl, and then add 37 ° C.
Heated for 2 hours.

FIG. 8A shows the results of 16% SDS-PAGE before and after heating. The samples after heating had β-galactosidase derivative, V8, which had molecular weights of 12 kDa, 26 kDa, and 22 kDa, respectively.
It can be seen that a band corresponding to a part of the protease derivative, aminoglucoside 3′-phosphotransferase, has appeared. On the other hand, FIG. 8B shows a strain deficient in OmpT protease W3110 M25 (Sugimura, K. (1987) Biochem. Biophy.
s. Res. Commun. 153, 753-759) as a host, the V8D fusion protein was expressed, and the same operation was performed on the obtained inclusion body. In this case, the above 3 bands are SD
Since it was not detected in S-PAGE, the processing of this fusion protein was performed using OmpT protease (Sugimura, K. and
Nishihara, T. (1988) J. Bacteriol., 170, 5625-5632
) Is considered to be a specific cleavage.

Furthermore, when a 26 kDa band was cut out from SDS-PAGE and the N-terminal amino acid sequence was examined, the R6 sequence (RLYR
(RHHRWGRSGSPLRAHE) (SEQ ID NO: 1) was confirmed to be cleaved at the center of the RR part, and the fusion protein was cleaved by Om.
It was confirmed that pT protease specifically performed. The above operation was carried out in the presence of 8 M urea when dissolving inclusion bodies and 4 M urea when processing. The above results indicate that OmpT protease is resistant to such high concentrations of urea and dissolves from inclusion bodies. It was shown for the first time that the fusion protein thus obtained can be specifically cleaved.

Example 6. Recombinant V8 protease (V8
The sample after the refolding processing of D) was diluted 20 times with 0.4 M potassium phosphate buffer (pH 7.5), and left overnight on ice.
Recombinant V8 protease (V8D) was refolded by this operation, and it was measured according to the above-mentioned method.
An activity corresponding to g / ml was obtained. The refolding efficiency was about 20%. FIG. 9 shows the results of 16% SDS-PAGE before and after the refolding operation.

When the recombinant V8 protease (V8D) is refolded, it acts as a strong protease to degrade β-galactosidase derivatives, aminoglucoside 3′-phosphotransferase-derived proteins, and other Escherichia coli-derived proteins. Therefore, the bands of those proteins disappear. Therefore, the sample after the refolding procedure is the recombinant V8 protease (V8D)
Only the major protein remains, which can be expected to greatly reduce the subsequent purification procedure. Recombinant V8 thus obtained
Protease (V8D) has a C-terminal deletion of 56 amino acids as compared to the natural type due to the construction of the gene, but the activity is maintained, and it was revealed that this deleted region is not essential for the activity. .

Example 7. Refolding from inclusion body
Group of recombinant V8 protease (V8D) obtained by
In order to compare the substrate specificity of recombinant V8 protease (V8D) obtained by cytospecific refolding with that of natural type, a fusion protein of human calcitonin precursor (hCT [G]) was used as a substrate, and the protein An experiment was conducted to release both hCT [G] and both proteases.

The human calcitonin fusion protein used in the experiment was a β-galactosidase derivative (108 amino acids) and hC.
It has a structure in which T [G] is fused via a linker having glutamic acid, and natural V8 protease cleaves the peptide bond on the carboxyl side of this glutamic acid residue to hC.
T [G] can be released. As a plasmid having a gene encoding the fusion protein, pG97S4
DhCT [G] R4 (see JP-A-5-328992) can be mentioned.

10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 5 mM DT
To 1 ml of a solution containing T, 2M urea and 10 mg / ml human calcitonin fusion protein, 1.2 μg of the recombinant V8 protease (V8D) equivalent to the activity of natural V8 protease was added, and the mixture was added at 30 ° C for 1 hour. After reacting for a period of time, the reaction solution was subjected to high performance liquid chromatography (0.1% trifluoroacetic acid (TF
A) and 0.1% TFA / 50% acetonitrile for elution with a linear concentration gradient).

As the recombinant V8 protease (V8D), a sample after refolding was directly used, and as a comparative control, a sample obtained by allowing a commercially available natural V8 protease to act under the same conditions was used. Figure 10 shows the elution pattern of high performance liquid chromatography. It was confirmed that the cleavage pattern for the human calcitonin fusion protein was the same between the recombinant V8 protease (V8D) and the native V8 protease, and that both proteases were equivalent in terms of substrate specificity.

Example 8. C-terminal side of V8 protease
In order to prepare a fusion protein in which the C-terminal side of the V8 protease region is extended by 2, 3, 4, 6 and 8 amino acids as compared with the V8D fusion protein, the PCR primers shown in FIG. 11 were synthesized. PV8F, which encodes a fusion protein of a type extended by 3 amino acids (V8F fusion protein), was prepared by the following method. Using the primer b and the primer I shown in Fig. 1 (b), pV8RPT (-) prepared in Example 1 was used as a template DNA.
An amplification reaction on the V8 protease gene side was performed using 1 μg, and then the fragment was cleaved with EcoRI and SacI to prepare a 0.1 kb gene fragment.

On the other hand, the amplification reaction of the R6 linker sequence and the aminoglucoside 3 ′ phosphotransferase gene part was carried out using primer g and primer h and 0.1 μg of pV8D as the template DNA, followed by digestion with EcoT22I and SacI.
A 0.3 kb gene fragment was prepared. The PCR was carried out according to the conditions of Example 1. 0.1kb and 0.3kb obtained in this way
Was ligated with the EcoRI-EcoT22I fragment (4.2 kb) of pV8D to prepare pV8F (see FIGS. 12 and 14). Primer a,
For c, d, e and primer g, the same operation (however,
(Use NdeI instead of SacI for the construction of pV8H)
By doing so, pV8H, pV8A, pV8D2 and pV8Q were prepared. The combinations of primers and template DNA used for the construction of these plasmids are shown below.

PV8H: primer a, primer I, pV8R
Prepared from the PCR products obtained by combining PT (-) with primer f, primer h, and pV8D. pV8A: prepared from PCR products obtained by combining primer c, primer I, pV8RPT (-) and primer g, primer h, pV8D. pV8D2: Prepared from PCR products obtained by combining primer d, primer I, pV8RPT (-) and primer g, primer h, pV8D. pV8Q: prepared from PCR products obtained from the combination of primer e, primer I, pV8RPT (-) and primer g, primer h, pV8D.

Compared with the V8D fusion protein shown in Example 4, these plasmids produced a fusion protein in which the C-terminal portion of the V8 protease region was extended by 2, 4, 6 and 8 amino acids, respectively (Fig. 13, see Fig. 14). JM
FIG. 13 shows the results of investigating the expression of each fusion protein by the same procedure as in Example 4 after transforming 101 strains with these plasmids. The formation of inclusion bodies was observed in pV8H, pV8F and pV8D, and the inclusion bodies thus obtained could be refolded into active recombinant V8 protease by the procedure shown in Example 5. On the other hand, in the case of pV8A, pV8D2 and pV8Q, inclusion bodies were not obtained and V8 protease activity was observed in the soluble fraction.

In the case of these plasmids, the expressed fusion protein has protease activity, and it is considered that the inhibition of proliferation is the cause of not forming inclusion bodies. That is, V8
In order to express the protease as an inactive fusion protein, it is important to fuse before the phenylalanine at the 215th position from the N-terminus, and when fused beyond this site, the V8 protease part forms the original three-dimensional structure. It was found that a fusion protein with protease activity is produced, which causes growth inhibition and cannot be highly expressed.

[0079]

INDUSTRIAL APPLICABILITY According to the present invention, a desired polypeptide can be produced in large quantities and at low cost. In particular, in the present invention, S. aureus V8 protease was investigated as the target polypeptide, but a high-purity enzyme was gene-recombinantly used, and a safe host cell such as Escherichia coli was used, whereby a large amount and inexpensive It has become possible to carry out efficient purification and manufacturing.

[0080]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Arg Leu Tyr Arg Arg His His Arg Trp Gly Arg Ser Gly Ser Pro Leu 5 10 15 Arg Ala His Glu 20

SEQ ID NO: 2 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence ACCGCTCGAG GTTATATTAC CAAATAACGA T 31

SEQ ID NO: 3 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CTTAATGTCG ACTTAAGCTG CATCTGGATT 30

SEQ ID NO: 4 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence TCGCGTCGAC TTATTGGTCA TCGTTGGCAA A 31

SEQ ID NO: 5 Sequence length: 344 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Met Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp 1 5 10 15 Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro 20 25 30 Pro Phe Ala Ser Trp Arg Asn Ser Asp Asp Ala Arg Thr Asp Arg Pro 35 40 45 Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe 50 55 60 Pro Ala Pro Glu Ala Val Pro Asp Ser Leu Leu Asp Ser Asp Leu Pro 65 70 75 80 Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr 85 90 95 Asp Ala Glu Leu Arg Leu Tyr Arg Arg His His Arg Trp Gly Arg Ser 100 105 110 Gly Ser Pro Leu Arg Ala His Glu Gln Phe Leu Glu Val Ile Leu Pro 115 120 125 Asn Asn Asp Arg His Gln Ile Thr Asp Thr Thr Asn Gly His Tyr Ala 130 135 140 Pro Val Thr Tyr Ile Gln Val Glu Ala Pro Thr Gly Thr Phe Ile Ala 145 150 155 160 Ser Gly Val Val Val Gly Lys Asp Thr Leu Leu Thr Asn Lys His Val 165 170 175 Val Asp Ala Thr His Gly Asp Pro His Ala Leu Lys Ala Phe Pro Ser 180 185 190 Ala Ile Asn Gln Asp Asn Tyr Pro Asn Gly Gly Phe Thr Ala Glu Asn 195 200 205 Ile Thr Lys Tyr Ser Gly Glu Gly Asp Leu Ala Ile Val Lys Phe Ser 210 215 220 Pro Asn Glu Gln Asn Lys His Ile Gly Glu Val Val Lys Pro Ala Thr 225 230 235 240 Met Ser Asn Asn Ala Glu Thr Gln Val Asn Gln Asn Ile Thr Val Thr 245 250 255 Gly Tyr Pro Gly Asp Lys Pro Val Ala Thr Met Trp Glu Ser Lys Gly 260 265 270 Lys Ile Thr Tyr Leu Lys Gly Glu Ala Met Gln Tyr Asp Leu Ser Thr 275 280 285 Thr Gly Gly Asn Ser Gly Ser Pro Val Phe Asn Glu Lys Asn Glu Val 290 295 300 Ile Gly Ile His Trp Gly Gly Val Pro Asn Glu Phe Asn Gly Ala Val 305 310 315 320 Phe Ile Asn Glu Asn Val Arg Asn Phe Leu Lys Gln Asn Ile Glu Asp 325 330 335 Ile His Phe Ala Asn Asp Asp Gln 340

SEQ ID NO: 6 Sequence length: 392 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Met Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp 1 5 10 15 Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro 20 25 30 Pro Phe Ala Ser Trp Arg Asn Ser Asp Asp Ala Arg Thr Asp Arg Pro 35 40 45 Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe 50 55 60 Pro Ala Pro Glu Ala Val Pro Asp Ser Leu Leu Asp Ser Asp Leu Pro 65 70 75 80 Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr 85 90 95 Asp Ala Glu Leu Arg Leu Tyr Arg Arg His His Arg Trp Gly Arg Ser 100 105 110 Gly Ser Pro Leu Arg Ala His Glu Gln Phe Leu Glu Val Ile Leu Pro 115 120 125 Asn Asn Asp Arg His Gln Ile Thr Asp Thr Thr Asn Gly His Tyr Ala 130 135 140 Pro Val Thr Tyr Ile Gln Val Glu Ala Pro Thr Gly Thr Phe Ile Ala 145 150 155 160 Ser Gly Val Val Val Gly Lys Asp Thr Leu Leu Thr Asn Lys His Val 165 170 175 Val Asp Ala Thr His Gly Asp Pro His Ala Leu Lys Ala Phe Pro Ser 180 185 190 Ala Ile Asn Gln Asp Asn Tyr Pro Asn Gly Gly Phe Thr Ala Glu Asn 195 200 205 Ile Thr Lys Tyr Ser Gly Glu Gly Asp Leu Ala Ile Val Lys Phe Ser 210 215 220 Pro Asn Glu Gln Asn Lys His Ile Gly Glu Val Val Lys Pro Ala Thr 225 230 235 240 Met Ser Asn Asn Ala Glu Thr Gln Val Asn Gln Asn Ile Thr Val Thr 245 250 255 Gly Tyr Pro Gly Asp Lys Pro Val Ala Thr Met Trp Glu Ser Lys Gly 260 265 270 Lys Ile Thr Tyr Leu Lys Gly Glu Ala Met Gln Tyr Asp Leu Ser Thr 275 280 285 Thr Gly Gly Asn Ser Gly Ser Pro Val Phe Asn Glu Lys Asn Glu Val 290 295 300 Ile Gly Ile His Trp Gly Gly Val Pro Asn Glu Phe Asn Gly Ala Val 305 310 315 320 Phe Ile Asn Glu Asn Val Arg Asn Phe Leu Lys Gln Asn Ile Glu Asp 325 330 335 Ile His Phe Ala Asn Asp Asp Gln Pro Asn Asn Pro Asp Asn Pro Asp 340 345 345 350 Asn Pro Asn Asn Pro Asp Asn Pro Asn Asn Pro Asp Glu Pro Asn Asn 355 360 365 Pro Asp Asn Pro Asn Asn Pro Asp Asn Pro Asp Asn Gly Asp Asn Asn 370 375 380 Asn Ser AspAsn Pro Asp Ala Ala 385 390

SEQ ID NO: 7 Sequence length: 532 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Met Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp 1 5 10 15 Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro 20 25 30 Pro Phe Ala Ser Trp Arg Asn Ser Asp Asp Ala Arg Thr Asp Arg Pro 35 40 45 Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe 50 55 60 Pro Ala Pro Glu Ala Val Pro Asp Ser Leu Leu Asp Ser Asp Leu Pro 65 70 75 80 Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr 85 90 95 Asp Ala Glu Leu Arg Leu Tyr Arg Arg His His Arg Trp Gly Arg Ser 100 105 110 Gly Ser Pro Leu Arg Ala His Glu Gln Phe Leu Glu Val Ile Leu Pro 115 120 125 Asn Asn Asp Arg His Gln Ile Thr Asp Thr Thr Asn Gly His Tyr Ala 130 135 140 Pro Val Thr Tyr Ile Gln Val Glu Ala Pro Thr Gly Thr Phe Ile Ala 145 150 155 160 Ser Gly Val Val Val Gly Lys Asp Thr Leu Leu Thr Asn Lys His Val 165 170 175 Val Asp Ala Thr His Gly Asp Pro His Ala Leu Lys Ala Phe Pro Ser 180 185 190 Ala Ile Asn Gln Asp Asn Tyr Pro Asn Gly Gly Phe Thr Ala Glu Asn 195 200 205 Ile Thr Lys Tyr Ser Gly Glu Gly Asp Leu Ala Ile Val Lys Phe Ser 210 215 220 Pro Asn Glu Gln Asn Lys His Ile Gly Glu Val Val Lys Pro Ala Thr 225 230 235 240 Met Ser Asn Asn Ala Glu Thr Gln Val Asn Gln Asn Ile Thr Val Thr 245 250 255 Gly Tyr Pro Gly Asp Lys Pro Val Ala Thr Met Trp Glu Ser Lys Gly 260 265 270 Lys Ile Thr Tyr Leu Lys Gly Glu Ala Met Gln Tyr Asp Leu Ser Thr 275 280 285 Thr Gly Gly Asn Ser Gly Ser Pro Val Phe Asn Glu Lys Asn Glu Val 290 295 300 Ile Gly Ile His Trp Gly Gly Val Pro Asn Glu Phe Asn Gly Ala Val 305 310 315 320 Phe Ile Asn Glu Asn Val Arg Asn Phe Leu Lys Gln Asn Ile Glu Asp 325 330 335 Arg Leu Tyr Arg Arg His His Arg Trp Gly Arg Ser Gly Ser Pro Leu 340 345 350 Arg Ala His Glu Gln Phe Leu Glu Cys Gly Asn Gly Lys Thr Ala Phe 355 360 365 Gln Val Leu Glu Glu Tyr Pro Asp Ser Gly Glu Asn Ile Val Asp Ala 370 375 380 Leu Ala ValPhe Leu Arg Arg Leu His Ser Ile Pro Val Cys Asn Cys 385 390 395 400 Pro Phe Asn Ser Asp Arg Val Phe Arg Leu Ala Gln Ala Gln Ser Arg 405 410 415 Met Asn Asn Gly Leu Val Asp Ala Ser Asp Phe Asp Asp Glu Arg Asn 420 425 430 Gly Trp Pro Val Glu Gln Val Trp Lys Glu Met His Lys Leu Leu Pro 435 440 445 Phe Ser Pro Asp Ser Val Val Thr His Gly Asp Phe Ser Leu Asp Asn 450 455 460 Leu Ile Phe Asp Glu Gly Lys Leu Ile Gly Gly Ile Asp Val Gly Arg 465 470 475 480 Val Gly Ile Ala Asp Arg Tyr Gln Asp Leu Ala Ile Leu Trp Asn Cys 485 490 495 Leu Gly Glu Phe Ser Pro Ser Leu Gln Lys Arg Leu Phe Gln Lys Tyr 500 505 510 Gly Ile Asp Asn Pro Asp Met Asn Lys Leu Gln Phe His Leu Met Leu 515 520 525 Asp Glu Phe Phe 530

SEQ ID NO: 8 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence ATCGTTGGCC ATATGGATAT CTTCAATATT 30

SEQ ID NO: 9 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GACTTATTGG TCATCGAGCT CAAAATGGAT ATC 33

SEQ ID NO: 10 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GACTTATTGG TCGAGCTCGG CAAAATGGAT 30

SEQ ID NO: 11 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence ATCTGGGTTG AGCTCATCGT TGGCAAAATG GAT 33

SEQ ID NO: 12 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence ATCTGGTTGG AGCTCTTGGT CATCGTTGGC AAA 33

SEQ ID NO: 13 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence ACAAAATCAT ATGGAACGCC TATATCGCCG ACAT 34

SEQ ID NO: 14 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence AATATTGAAG AGCTCCGCCT ATATCGCCGA CAT 33

SEQ ID NO: 15 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GAATGGCAAA AGCTTATGCA TTTCTTT 27

SEQ ID NO: 16 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Asn Ile Glu Asp Arg Leu Tyr Arg Arg His His Arg 5 10

SEQ ID NO: 17 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Asn Ile Glu Asp Ile His Met Glu Arg Leu Tyr Arg Arg His His Arg 5 10 15

SEQ ID NO: 18 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Asn Ile Glu Asp Ile His Phe Glu Leu Arg Leu Tyr Arg Arg His His 5 10 15 Arg

SEQ ID NO: 19 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Asn Ile Glu Asp Ile His Phe Ala Glu Leu Arg Leu Tyr Arg Arg His 5 10 15 His Arg

SEQ ID NO: 20 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Asn Ile Glu Asp Ile His Phe Ala Asn Asp Glu Leu Arg Leu Tyr Arg 5 10 15 Arg His His Arg 20

SEQ ID NO: 21 Sequence length: 22 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Asn Ile Glu Asp Ile His Phe Ala Asn Asp Asp Gln Glu Leu Arg Leu 5 10 15 Tyr Arg Arg His His Arg 20

SEQ ID NO: 22 Sequence length: 213 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Val Ile Leu Pro Asn Asn Asp Arg His Gln Ile Thr Asp Thr Thr Asn 1 5 10 15 Gly His Tyr Ala Pro Val Thr Tyr Ile Gln Val Glu Ala Pro Thr Gly 20 25 30 Thr Phe Ile Ala Ser Gly Val Val Val Gly Lys Asp Thr Leu Leu Thr 35 40 45 Asn Lys His Val Val Asp Ala Thr His Gly Asp Pro His Ala Leu Lys 50 55 60 Ala Phe Pro Ser Ala Ile Asn Gln Asp Asn Tyr Pro Asn Gly Gly Phe 65 70 75 80 Thr Ala Glu Asn Ile Thr Lys Tyr Ser Gly Glu Gly Asp Leu Ala Ile 85 90 95 Val Lys Phe Ser Pro Asn Glu Gln Asn Lys His Ile Gly Glu Val Val 100 105 110 Lys Pro Ala Thr Met Ser Asn Asn Ala Glu Thr Gln Val Asn Gln Asn 115 120 125 Ile Thr Val Thr Gly Tyr Pro Gly Asp Lys Pro Val Ala Thr Met Trp 130 135 140 Glu Ser Lys Gly Lys Ile Thr Tyr Leu Lys Gly Glu Ala Met Gln Tyr 145 150 155 160 Asp Leu Ser Thr Thr Gly Gly Asn Ser Gly Ser Pro Val Phe Asn Glu 165 170 175 Lys A sn Glu Val Ile Gly Ile His Trp Gly Gly Val Pro Asn Glu Phe 180 185 190 Asn Gly Ala Val Phe Ile Asn Glu Asn Val Arg Asn Phe Leu Lys Gln 195 200 205 Asn Ile Glu Asp Ile 210

SEQ ID NO: 23 Sequence length: 214 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Val Ile Leu Pro Asn Asn Asp Arg His Gln Ile Thr Asp Thr Thr Asn 1 5 10 15 Gly His Tyr Ala Pro Val Thr Tyr Ile Gln Val Glu Ala Pro Thr Gly 20 25 30 Thr Phe Ile Ala Ser Gly Val Val Val Gly Lys Asp Thr Leu Leu Thr 35 40 45 Asn Lys His Val Val Asp Ala Thr His Gly Asp Pro His Ala Leu Lys 50 55 60 Ala Phe Pro Ser Ala Ile Asn Gln Asp Asn Tyr Pro Asn Gly Gly Phe 65 70 75 80 Thr Ala Glu Asn Ile Thr Lys Tyr Ser Gly Glu Gly Asp Leu Ala Ile 85 90 95 Val Lys Phe Ser Pro Asn Glu Gln Asn Lys His Ile Gly Glu Val Val 100 105 110 Lys Pro Ala Thr Met Ser Asn Asn Ala Glu Thr Gln Val Asn Gln Asn 115 120 125 Ile Thr Val Thr Gly Tyr Pro Gly Asp Lys Pro Val Ala Thr Met Trp 130 135 140 Glu Ser Lys Gly Lys Ile Thr Tyr Leu Lys Gly Glu Ala Met Gln Tyr 145 150 155 160 Asp Leu Ser Thr Thr Gly Gly Asn Ser Gly Ser Pro Val Phe Asn Glu 165 170 175 Lys As n Glu Val Ile Gly Ile His Trp Gly Gly Val Pro Asn Glu Phe 180 185 190 Asn Gly Ala Val Phe Ile Asn Glu Asn Val Arg Asn Phe Leu Lys Gln 195 200 205 Asn Ile Glu Asp Ile His 210

SEQ ID NO: 24 Sequence length: 215 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide sequence Val Ile Leu Pro Asn Asn Asp Arg His Gln Ile Thr Asp Thr Thr Asn 1 5 10 15 Gly His Tyr Ala Pro Val Thr Tyr Ile Gln Val Glu Ala Pro Thr Gly 20 25 30 Thr Phe Ile Ala Ser Gly Val Val Val Gly Lys Asp Thr Leu Leu Thr 35 40 45 Asn Lys His Val Val Asp Ala Thr His Gly Asp Pro His Ala Leu Lys 50 55 60 Ala Phe Pro Ser Ala Ile Asn Gln Asp Asn Tyr Pro Asn Gly Gly Phe 65 70 75 80 Thr Ala Glu Asn Ile Thr Lys Tyr Ser Gly Glu Gly Asp Leu Ala Ile 85 90 95 Val Lys Phe Ser Pro Asn Glu Gln Asn Lys His Ile Gly Glu Val Val 100 105 110 Lys Pro Ala Thr Met Ser Asn Asn Ala Glu Thr Gln Val Asn Gln Asn 115 120 125 Ile Thr Val Thr Gly Tyr Pro Gly Asp Lys Pro Val Ala Thr Met Trp 130 135 140 Glu Ser Lys Gly Lys Ile Thr Tyr Leu Lys Gly Glu Ala Met Gln Tyr 145 150 155 160 Asp Leu Ser Thr Thr Gly Gly Asn Ser Gly Ser Pro Val Phe Asn Glu 165 170 175 Lys As n Glu Val Ile Gly Ile His Trp Gly Gly Val Pro Asn Glu Phe 180 185 190 Asn Gly Ala Val Phe Ile Asn Glu Asn Val Arg Asn Phe Leu Lys Gln 195 200 205 Asn Ile Glu Asp Ile His Phe 210 215

[Brief description of drawings]

FIG. 1 shows Staphylococcus aureus ( St.
aphylococcus aureus ) V8 protease gene constitution (a) and P used for cloning the gene of the present invention
It is a figure which shows the base sequence (b) of the primer for CR.

FIG. 2 shows pG97S4DhCT [G] R6 and pG97S4DhCT [G] R.
FIG. 8 is a diagram showing a manufacturing process of 10.

FIG. 3 shows plasmids pV8RPT (+) and pV8RPT.
It is a figure which shows the manufacturing process of (-).

FIG. 4 shows plasmids pV8RPT (+) and pV8RPT.
It is a figure which shows the amino acid sequence of the fusion protein encoded by (-).

FIG. 5 is a diagram showing a production process of plasmid pV8D.

FIG. 6 shows the amino acid sequence of the fusion protein encoded in plasmid pV8D.

FIG. 7 is an electrophoretogram showing that the fusion protein of the present invention forms inclusion bodies and migrates to the insoluble fraction.
It is a photograph instead of a drawing.

FIG. 8 is an electrophoretogram showing that the V8D fusion protein is cleaved by the host-derived protease ompT to release V8 protease, and is a photograph as a drawing.

FIG. 9: V8 released by ompT protease
It is an electropherogram which shows the refolding of a protease, and is a photograph instead of a drawing.

FIG. 10 shows that a fusion protein containing a human calcitonin precursor was obtained by using the recombinant V8 protease (A) or S. cerevisiae obtained by the method of the present invention. FIG. 7 is a chart comparing products when cleaved by V8 protease (B) from Aureus.

FIG. 11: DNs encoding various fusion proteins
A-containing plasmids (pV8H, pV8F, pV8A, pV8D2 and pV
It is a figure which shows the base sequence of the primer used in preparation of 8Q).

FIG. 12 shows plasmids pV8H, pV8F, pV8A, pV.
It is a figure which shows the process of preparation of 8D2 and pV8Q.

FIG. 13 shows plasmids pV8D, pV8H, pV8F and pV.
It is the figure which displayed the C-terminal amino acid sequence of V8 protease encoded in 8A, pV8D2, and pV8Q, and the presence or absence of the formation of the inclusion body by the expression product (fusion protein) of each plasmid.

FIG. 14 shows an example of an amino acid sequence of a target polypeptide produced by the method of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 15/09 ZNA // (C12P 21/02 C C12R 1:19) (C12N 9/52 C12R 1 : 19) (C12N 15/09 ZNA C12R 1: 445) C12R 1: 445)

Claims (34)

[Claims] 1. A host cell is transformed with an expression vector having a gene encoding a fusion protein comprising a protected polypeptide and a target polypeptide, the gene is expressed, and the fusion protein is expressed by an endogenous protease of the host cell. A method for producing a target polypeptide, which comprises cleaving the target polypeptide. 2. The fusion protein is represented by the formula (1) A-L-B or the formula (2) A-L-B-L-C (A and C are protected polypeptides, B is a target polypeptide, and L is a host. A linker peptide having a substrate which is specifically recognized by an intracellular endogenous protease), and the fusion protein is cleaved at the region of the linker peptide L to obtain the target polypeptide B from the fusion protein. The manufacturing method according to claim 1. 3. A target protein is expressed as an inactive fusion protein in a host cell, the cells are crushed, the fusion protein is separated, and the fusion protein is solubilized with a denaturing agent that solubilizes the fusion protein. 3. The method according to claim 1, wherein the target polypeptide is obtained from the fusion protein by solubilizing and then cleaving the linker peptide L region with a host cell endogenous protease. 4. A target polypeptide is expressed as an inactive fusion protein in a host cell, the cells are disrupted to separate the fusion protein, and then the fusion protein is solubilized with a denaturing agent that solubilizes the fusion protein. 4. Then, the target polypeptide is obtained from the fusion protein by lowering the concentration of the denaturing agent and then cleaving the linker peptide L with a host cell endogenous protease. 5. The method according to any one of claims 1 to 4, wherein the host cell endogenous protease and the fusion protein are present in the same fraction in the separation operation after disruption of the cells. 6. The host cell endogenous protease is Escherichia coli om
It is pT protease, The manufacturing method of any one of Claims 1-5 characterized by the above-mentioned. 7. The production method according to claim 3, wherein the denaturant for solubilizing the fusion protein is urea, guanidine hydrochloride or a surfactant. 8. The method according to claim 7, wherein the denaturant for solubilizing the fusion protein is urea and the urea concentration is 1-6M. 9. The host cell endogenous protease is Escherichia coli om.
9. The method according to any one of claims 3 to 8, wherein the method is pT protease, and the concentration of urea in the solution when the fusion protein is cleaved using the enzyme is 1-6M. 10. The linker peptide according to claim 2, which is a linker peptide having a site that is specifically recognized by an endogenous protease of a host cell having an expression vector of the target polypeptide. 1
The manufacturing method according to item. 11. The linker peptide is a peptide comprising 1 to 2 basic amino acid pairs in the linker peptide, and the linker peptide is composed of 2 to 50 amino acid residues. 10. The manufacturing method according to any one of 10. 12. The linker peptide according to claim 2, wherein the linker peptide contains 1 to 2 basic amino acid pairs at the N-terminal portion and / or C-terminal portion of the linker peptide. Production method. 13. The linker peptide has an amino acid sequence RL.
The method according to claim 11 or 12, which comprises YRRHHRWGRSGSPLRAHE (SEQ ID NO: 1). 14. The protected polypeptide is an Escherichia coli β-galactosidase-derived polypeptide and / or an aminoglycoside 3′-phosphotransferase-derived polypeptide derived from transposon 903, according to any one of claims 1 to 13. The manufacturing method described in. 15. The protected polypeptide A is an Escherichia coli β-galactosidase-derived polypeptide, and the protected polypeptide C is an aminoglycoside 3′-phosphotransferase-derived polypeptide derived from transposon 903. 14. The manufacturing method according to any one of 14. 16. A method for producing a target polypeptide, comprising:
The following production steps; (1) a step of causing refolding so that the target polypeptide becomes an active form, and the production method according to any one of claims 1 to 15. 17. A target polypeptide cleaved by ompT protease in the presence of a denaturant is refolded by decreasing the concentration of the denaturant to obtain an active target polypeptide. Item 6
17. The manufacturing method according to any one of items 1 to 16. 18. A polypeptide of interest comprises 20 to 800 amino acid residues.
The manufacturing method according to any one of 1. 19. The method according to claim 1, wherein the target polypeptide is a physiologically active polypeptide. 20. The physiologically active polypeptide is motilin, glucagon, adrenocortical hormone, adrenocorticotropic hormone releasing hormone, secretin, growth hormone, insulin, growth hormone secretory hormone, pazopressin,
Oxytocin, gastrin, glucagon-like peptide (G
LP-1), glucagon-like peptide (GLP-2), glucagon-like peptide (7-36 amide), cholecystokinin, vasoactive intestinal polypeptide (VIP), pituitary adenolate cycler Activating polypeptide (Pi
tuitary adenolate cyclase activating polypeptid
e), gastrin-releasing hormone, galanin, thyroid-stimulating hormone, luteinizing hormone-releasing hormone, calcitonin, parathyroid hormone (PTH, PTH (1-34) P
TH (1-84)), peptide histidine isoleucine (PHI), neuropeptide Y (neuropeptide Y), peptide YY (pept)
ide YY), pancreatic polypeptide (pancreat)
ic polypeptide), somatostatin, TGF-α, TG
The method according to claim 19, which is F-β, nerve growth factor, fibroblast growth factor, relaxin, prolactin, diuretic peptide, angiotensin, brain-derived neurotrophic factor (BDNF), or KEX2 endoprotease. 21. The diuretic peptide is ANP, BNP
21. The method of claim 20, which is or CNP. 22. The method according to claim 19, wherein the target polypeptide is an enzyme. 23. The method according to claim 22, wherein the target polypeptide is a proteolytic enzyme. 24. The method according to claim 23, wherein the enzyme is a KEX2 endoprotease or the protease derivative. 25. The method according to claim 23, wherein the target polypeptide is Staphylococcus aureus V8 protease or a protease derivative thereof. 26. The target polypeptide is a Staphylococcus aureus V8 protease derivative having the amino acid sequence shown in the underlined portion of FIG. 4 or 6 or the amino acid sequence shown in FIG. 14. 26. The manufacturing method according to claim 25, wherein 27. A method for producing a target polypeptide using gene recombination technology, comprising the following production steps: (1) The fusion protein comprises a protected polypeptide, a target polypeptide and a linker peptide, and the protected polypeptide. Is a β-galactosidase-derived polypeptide derived from Escherichia coli and / or an aminoglycoside 3′-phosphotransferase-derived polypeptide derived from transposon 903, and the target polypeptide is a Staphylococcus aureus V8 protease derivative, and The expression vector having a gene encoding the fusion protein, in which the linker peptide region between the protected polypeptide and the polypeptide of interest is a linker peptide region having a substrate specifically recognized by a host cell endogenous protease, E. coli is a vesicle transformed to express the fusion protein in E. coli with (2) Staphylococcus aureus (Staphylococcus aureus) V8 protease derivative is inactive states,
(3) After crushing the cells and separating the fusion protein,
A fraction containing the E. coli ompT protease, which is an intracellular protease, and the fusion protein is present, (4) solubilizing the fusion protein with a denaturant, and (5) solubilizing the E. coli om.
The step of obtaining the desired polypeptide from the fusion protein by lowering the concentration of the denaturant until the activity of the pT protease is expressed, and cleaving the linker peptide region with the protease, Peptide manufacturing method. 28. The denaturant for solubilizing the fusion protein is urea,
28. The method according to claim 27, which is guanidine hydrochloride or a surfactant. 29. The method according to claim 28, wherein the denaturant for solubilizing the fusion protein is urea and the urea concentration is 1-8M. 30. The method according to any one of claims 27 to 29, wherein the concentration of urea in the processing step with E. coli ompT protease is about 4M. 31. The linker peptide is a peptide comprising a dibasic amino acid pair at the N-terminus and C-terminus of the linker peptide, or in the linker peptide, and the linker peptide consisting of 2 to 50 amino acid residues. 31. The manufacturing method according to claim 27, wherein 32. The linker peptide has the amino acid sequence RL.
32. The method according to claim 27, which comprises YRRHHRWGRSGSPLRAHE (SEQ ID NO: 1). 33. The polypeptide of interest is any of Staphylococcus aureus V8 protease derivatives having the amino acid sequence shown in the underlined portion of FIG. 4 or 6 or the amino acid sequence shown in FIG. 14. 33. The manufacturing method according to claim 27, wherein: 34. A method for producing a target polypeptide using a gene recombination technique, which comprises the following production steps; (1) a step of causing refolding so that the target polypeptide is in an active form. 34. The manufacturing method according to claim 27, wherein: