WO1996002661A1 - Process for producing protein capable of molecular weight reduction caused by protease of host-cell origin - Google Patents

Abstract

A process for producing a protein capable of molecular weight reduction caused by a protease of host-cell origin, which comprises culturing a host cell which can produce and secrete such a protein in at least one medium selected from the group consisting of acidic amino acids, basic amino acids, phosphate salts, ammonium salts and nonionic surfactants and separating the target protein from the supernatant of the medium. Another process for producing the above protein comprises separating the target protein by treating the supernatant with a buffer containing an ammonium salt in a column chromatograph. The invention also relates to a yeast of the genus Pichia which secretes and expresses prourokinase. This process permits the mass production in the complete form of a protein capable of molecular weight reduction caused by a protease produced by genetic engineering techniques or fermentation. It can, in addition, increase host cell growth and production of the above protein and can accelerate protein secretion from the host cell. Further, the invention process can effectively control the reduction in the molecular weight of the protein secreted in the supernatant.

Description

 Specification

 PROCESS FOR PRODUCING PROTEIN LOWER MOLECULAR BY HOST CELL PROTEASE

 Technical field

 The present invention relates to a method for stably producing a large amount of an unstable protein which can be reduced in molecular weight by a protease derived from a host cell. Specifically, host cells that produce and secrete the protein obtained by genetic manipulation are cultured in a medium containing a specific substance to enhance the growth of the host cells and promote the production and secretion of the protein, and The present invention relates to a method for mass-producing a protein that can be reduced in molecular weight by a host cell-derived lipase by suppressing the reduction in molecular weight.

 Background art

 Known plasminogen activators involved in dialysis are tissue t-PA produced by vascular endothelial cells and perokinase (UK). However, perokinase, when used in large quantities, has the drawback of inducing the degradation and activation of coagulation and fibrinolytic factors and inducing a tendency to bleed.

 In contrast, the present inventors have proposed an inactive precursor of human ostium kinase produced by human cells [hereinafter referred to as blow mouth kinase. Japanese Unexamined Patent Publication (Kokai) No. 60-62981 (EP-B-1 394 47), J. Biol. Chem .. 260, 12 377 (1985)] is a fiber constituting a thrombus. It has a specific affinity for fibrin, has thrombolytic properties such as the selective degradation of fibrin, and more preferably, the blow mouth kinase, unlike perokinase, causes a bleeding tendency. Cell Struc. Func .. 10, 151 (1985)] found no defect.

 Therefore, the blow mouth kinase having such excellent properties is expected to be widely used clinically as a fibrinolytic enzyme.

 Under these circumstances, in recent years, attempts have been made to produce useful proteins such as blow mouth kinase in large quantities by using gene recombination technology. In particular, production using a recombinant (yeast) having a secretory system has been studied in various ways because of the advantage that the produced protein is simple and easy to purify.

However, when Sccharomyces cereuisiae is used as the host cell, And it is reported that mass production is difficult [Gene, 99, 235-241, 1991, J. Biol. Chem .. 265, (2), 801-807, 1990].

 In addition, the present inventors have used Pichia pastoris, which has an effect of increasing the secretion amount in the case of human serum albumin or invertase, as a host cell (Bio / Technology, 5, 1305-1308. 1985). Although the oral kinase was examined in the same manner, the secreted amount of the protein into the medium was lower than that of human serum albumin and the like, and considerable brourokinase remained in the cells without being secreted.

 Furthermore, the present inventors have found that when the Pichia yeast is cultured in a YPM medium, there are many degradants of about 47 kDa and about 30 kDa in addition to those of 5 O kDa which are considered to be intact proteins in the culture. I noticed that This 47 kDa protein was determined to be a deletion of the intact blow-mouth kinase N-terminus from the results of ゥ -esten blotting with a monoclonal antibody recognizing the EGF domain (Evider-Malgro-factor). However, it was considered that the Blow-mouth kinase was reduced in molecular weight by proteases derived from host cells and the like present in the culture supernatant.

 In addition, like Blow Mouth Kinase, there are also known proteins whose molecular weight is reduced by proteases derived from host cells and which is difficult to mass-produce by recombinant DNA technology (mouse EGF: Clare JI et al., Gene. 105, 205-212. 1991; Human EGF: PC D International Publication No. WO9003341; / S-endolphin: Bitter. GA et al., Proc. Natl. Acad. Sci. USA .. 81, 5330. 1984, etc. ).

 Therefore, in order to commercialize such a useful protein in various fields including the clinical field, it is desired to solve the above problems and establish a method for producing a useful protein by recombinant DNA technology. I have.

 Disclosure of the invention

Under such technical background, the present invention relates to the production of a protein that can be reduced in molecular weight by a protease derived from a host cell by an enzymatic or genetic engineering technique, wherein the protein is reduced in molecular weight. It is an object of the present invention to provide a method for mass production without mass production. Specifically, a host cell-derived protein that can be degraded by a host cell-derived protease is added to the host cell. It is an object of the present invention to provide a method for producing a large S protein by stably producing and secreting a large amount of white matter and suppressing the low molecular weight after secretion. The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that the recombinant host cell is at least one selected from acidic amino acids, basic amino acids, phosphates, ammonium salts and nonionic surfactants. By culturing in a seed-containing medium, a large amount of protein that can be degraded by a host cell-derived protease is produced and secreted, and the degrading by the protease in the medium is suppressed. I found that. Furthermore, by treating the culture supernatant with column chromatography in the presence of ammonium salt, the lowering of the molecular weight of the target protein is effectively suppressed, and a large amount of the target protein can be isolated from the culture supernatant. And completed the present invention. That is, the present invention relates to a host cell that secretes and produces a protein that can be reduced in molecular weight by a protease derived from a host cell, and more particularly, to a method for transforming Pichia yeast into an acidic amino acid, a basic amino acid, a phosphate, an ammonium salt, and a nonionic interface. Culturing in a medium containing at least one selected from the group consisting of activators, and collecting the protein from the resulting culture supernatant. How to make protein

 Further, the present invention provides a method for collecting a protein which can be degraded by a protease derived from a host cell from a culture supernatant in the production method, the method comprising the steps of: using a buffer solution containing ammonium salt for column chromatography on a culture supernatant; A method for producing a protein that can be reduced in molecular weight with a host cell-derived protease, which comprises treating with a hydrophobic column chromatography and a cation exchange column chromatography, preferably.

 Furthermore, the present invention provides a transformant (Bikia yeast) that produces and secretes blow mouth kinase.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing a method of constructing the plasmids p K O 110, ρ Ο 1 113, and ρ Κ Ο 114.

FIG. 2 shows a method for constructing plasmids pKO116 and pKO117. ₀

 FIG. 3 is a diagram showing a method for constructing brassmid pKO122 and a blow-mouth kinase secretion expression vector ρΚΟ126.

 FIG. 4 is a diagram showing a restriction enzyme map of the blow mouth kinase secretion expression vector pKO126.

 FIG. 5 is a graph showing the growth of Pichia yeast SC06 strain cultured in five types of methanol media in Experimental Example 1 over time. 〇: Medium 1, medium: Medium 2, □: Medium 3, X: Medium 4, ·: Medium 5

 FIG. 6 is a diagram showing the time course of the Α activity in the culture supernatant obtained by culturing the Bikia yeast SC06 strain in five types of methanol media in Experimental Example 1. 〇: Medium 1, Medium: Medium 2, □: Medium 3, X: Medium 4, ·: Medium 5

 FIG. 7 shows the results of a Western plot (electrophoresis) performed in Experimental Example 4. Lane 1: Using ΥΡΜι medium

Lane 2: YP ₂ + 2¾NH ₄ H ₂ P0 ₄ + 2¾ (NH *) ₂ HP0 ₄

Lanes 3 and 4: ΥΡΜί + 0.01¾ Tri-ton X-100 + 0.1M arginine lanes 5 and 6: ΥΡΜ ₂ ten 0.01¾ Tri-ton X-100 + 0.1M arginine

+ 2¾ΝΗ ₄ Η ₂ Ρ04 + 2¾ (NH ₄ ) ₂ HP0 ₄

 Detailed description of the invention

 As used herein, the term "host cell-derived protease" refers to a protease encoded by a chromosome gene of a host cell. Such proteases are presently found in yeast in about 30 or more species, and are present in vacuoles (yscA, yscB, yscY), mitochondria (yscMpI), periplasm (ysdl) and the like. In cells, it is known to be involved in protein degradation, nitrogen metabolism, precursor processing, and the like (Hirsch, H. H. et al., Molecular and Cell Biology of Yeasts, p. 134-200. 1989).

The protein targeted by the production method of the present invention is not particularly limited as long as it can be reduced in molecular weight by the action of the above-described host cell-derived protease. Specifically, plow-mouth kinase, human tissue plasminogen activator, blood coagulation factor IX, blood coagulation factor X, protein c, interferon, epidermal grosspha Kuta-1 (hereinafter referred to as EGF) and ^ -dolphin are exemplified. Particularly preferred is blow mouth kinase.

 Further, the protein targeted by the present invention is not limited to a naturally-derived protein, and may be a mutant obtained by genetically mutating a natural protein or a derivative obtained by artificially modifying the natural protein. For example, in the case of blow-mouth kinase, the entire region or part of the evidermal macroglobulin factor domain of the natural blow-mouth kinase is deleted, or the entire region or part thereof is S-replaced with another amino acid residue. Or a protein obtained by deleting the entire region or a part of the finger domain of bloomlokinase, or substituting the entire region or a part thereof with another amino acid residue. 87180 (EP-A-398362), JP-A-3-87181 (EP-A-398361, U.S. Pat.No. 5,098,840), JP-A-5-957786 Gazette (EP—A—54 1952, U.S. Pat. No. 538 9538)]. More specifically, a blow-mouth kinase derivative in which the asparagine residue at position 302 from the N-terminus of the natural blow-mouth kinase is substituted with a glutamic acid residue, and an annexin having thrombus affinity at the C-terminal side of the natural blow-mouth kinase And a fusion protein to which V protein is bound (PCT International Publication No. WO 92/19279). These derivatives are sugar-free blow-mouth kinases to which no asparagine-linked sugar chain is added, and are expected to be applied as thrombolytic agents due to their high thrombus affinity.

 The host cell used in the present invention is not particularly limited as long as it can secrete and produce a protein that can be reduced in molecular weight by the above-mentioned protease, and it does not matter whether the host cell is naturally derived or obtained by gene manipulation. Absent. In addition to those already described in publicly known documents, those that will be developed in the future can be used as appropriate. Specifically, a bacterium having a function of expressing a gene encoding a protein that can be degraded by a host cell-derived protease in a cell, producing the protein, and secreting the protein outside the cell. , Escherichia coli, yeast, mold and Bacillus subtilis.

Specifically, it is a yeast, especially a genus Pichia or Saccharomyces. More specifically, Pichia pastoris (Pichi na /) a5toris) GTS 1 15 (his 4) (NRRL accession number Y—1 585 1) Etc. are exemplified. 方法 A method for preparing a host cell capable of secreting and producing a protein that can be reduced in molecular weight by the above protease by a gene recombination operation can be carried out by employing a known method or a method analogous thereto. For example, as a method for preparing a host capable of producing and secreting blow mouth kinase, a method described in JP-A-60-180591 (EP-B-154272) (Saccharomyces 厲), a human tissue plasminogen activator Examples of the method for producing a host that produces and secrete, include the method described in Transfusion Medicine, p303-313. (1986).

 When Pichia yeast is used as a host cell, it can be prepared by contacting the S gene of the target protein with a promoter of the AOX gene that is strongly induced by methanol in the medium according to a conventional method. In this case, both the autonomous propagation type and the chromosome integration type are used as the expression vector, but the expression vector is preferably the chromosome integration type. More preferably, the expression vector has the 5 'and 3' untranslated regions of the AOX gene at both ends, and is HIS4 which is one of the best candidates in addition to the AOX promoter, foreign gene and AOX terminator. It is a straight-type vector with a distant element. Transformation was performed by the spheroblast method (Cregg, JM et al., Mol. Cell. Biol .. 5.3376-3385, 1983), the alkali cation method (Ito, H .. et al., J. Bacterid .. 153, (1) 163). -168. 1983) can be used.

 In addition, a strain having histidine auxotrophy (his-) is preferably used as a host cell. By using such a strain, whether or not the expression vector has been introduced can be easily known. That is, the host cell into which the vector containing the H1S4 gene has been equally introduced loses its histidine auxotrophy and can be used as a cloning instruction.

 Until now, the secretory expression system of Pichia yeast is Inv e r t a se

 (Tschopp. JF., Et al., Bio / Technology, 5, 1305-1308, 1987), mouse EGF (Clare. JJ, et al., Gene. 105, 205-212, 1991), pencilisozyme (Digan,. E, et al., Bio / Technology, 7. 160-164. 1989), but no report on properokinase.

The production method of the present invention relates to a protein that can be reduced in molecular weight by a host cell-derived protease. Host cells capable of secreting and producing white matter are cultured in a medium containing at least one selected from the group consisting of acidic amino acids, basic amino acids, phosphates, ammonium salts, and nonionic surfactants. And This makes it possible to obtain a large amount of a protein which can be reduced in molecular weight by a host cell-derived porcine protease from the culture supernatant in an intact state. That is, the culture method is considered to contribute to expression of the target protein in host cells, improvement of production efficiency, promotion of extracellular secretion, or suppression of low molecular weight by protease.

 Examples of the acidic amino acid used in the present invention include aspartic acid and glutamic acid, and examples of the basic amino acid include arginine and lysine. Preferably, it is arginine or glutamic acid. Acidic amino acids and basic amino acids may be used in the form of a salt. Examples include sodium aspartate, potassium aspartate, sodium glutamate, potassium glutamate, alginine hydrochloride, and lysine hydrochloride.

 In addition, one or a combination of several of the above-described amino acids can be used. The amino acid content fi in the medium is usually about 0.02 to 0.5M, preferably about 0.05 to 0.3M, and more preferably about 0.1M.

By culturing the host cell of the present invention in a medium containing the amino acid, the effect of promoting the growth of bacterial cells and suppressing the reduction of the molecular weight of the produced protein can be obtained. Examples of the phosphate used in the present invention include ammonium phosphate I NHJ HPO * or NH ₄ H ₂ PO «] and sodium phosphate [Na ₂ HPO4 or

NaH ₂ PO *] and the like.

 The content of phosphate in the medium is about 2 to 5 w / v%, preferably about 4 w / v%.

As the Anmoniumu salt used in the present invention, phosphoric acid Anmoniu厶[(NH) ₂ HPO * or NH «H ₂ PO4], Anmoniumu carbonate [(NH _{4) 2} C0 ₃ or NH ₄ HCO3], Anmoniumu chloride [ NH ₄ Cl] and the like. The content of ammonium salt in the medium is about 2 to 5 wZv%, preferably about 4 wZv%. The phosphate or ammonium salt is preferably in the form of ammonium phosphate, more preferably in the form of a combination of (NH ₄ ) 2 HPO * and NH ₄ H ₂ PO ₄ . In the medium (NH *) 2~5w / v% is a 2 HPO * and NH ₄ H ₂ PO * content in total, and preferably about 4wZv ¾.

Li down黢塩or Anmoniumu salts, especially (NH ₄₎ 2 HPO4 and ΝΗ * Η ₂ Ρ0 by培蹇the host cell of the present invention ₄ in a medium containing, Protea is產生secreted from the host cell Ichize Thus, the effect of effectively suppressing the reduction of the molecular weight of a protein that can be reduced to a lower molecular weight is obtained.

 Examples of the nonionic surfactant used in the present invention include polyoxyethylene mono ρ-tert-butyl tert-butyl ether and polyoxyethylene sorbin fatty acid ester. Preferably, polyoxyethylene (9 to 10) mono-P-tert-octylphenyl ether (trade name: Triton X-100, Sigma), boroxyethylene (9) mono-p-tert-octylphenyl ether (Trade name Nonidet P-40, Shell), polyoxyethylene (20) sorbitan monolaurate (trade name Tween 20, Sigma), polyoxyethylene (20) sorbitan monooleate (trade name Tween 80, Sigma) And the like, with Triton X-100 being particularly preferred. The content of the nonionic surfactant in the medium is about 0.005 to 0.03 wZv%, preferably 0.01%.

 It is about.

The medium used in the present invention may contain at least one selected from the group consisting of the above acidic amino acids, basic amino acids, phosphates, ammonium salts and nonionic surfactants. Preferably, an embodiment containing (a) at least one selected from acidic amino acids and basic amino acids,) at least one selected from phosphates and ammonium salts, and) a nonionic surfactant. It is. Arginine in Naka preferably contains Anmoniumu phosphate [(NHJaHPO * and NH4H ₂ PO «] and 3-ton X- 1 00.

The other components are not particularly limited as long as the medium used in the present invention fulfills the above conditions and can grow and proliferate the host cells. Other combinations Examples of the component include components contained in a known medium commonly used in this field. For example, various sugars (glucose, glycerol, etc.) are generally used as carbon sources, urea and usate are used as nitrogen sources, various vitamins and nucleotides are used as sss nutrients, and inorganic salts such as Mg, Ca. Fe, Na, K , Mn, Co, Cu salts and the like.

 Specifically, YNB liquid medium [0.7% amino acid-free yeast nitrogen base (manufactured by Difco), 2% glucose], YPD liquid medium [1 〔yeast extract (manufactured by Diico), 2% bactopeptone (Difco) Co., Ltd., 2% glucose], etc. In addition, when the host cell is a methanol-assimilating host, a methanol-containing medium can be used. Is about 0.01 to 5%, and specifically, a YPM medium [1% yeast extract, 2 pact peptone, 0.01 to 5% methanol] is exemplified.

 Therefore, the medium used in the present invention is prepared by adding at least one selected from the above-mentioned acidic amino acids, basic amino acids, phosphates, ammonium salts and nonionic surfactants to a conventionally known medium. Can be easily prepared.

 The pH of the medium may be neutral, weakly basic or weakly acidic. It is preferably pH 6-8.

 The cultivation can be carried out by using the above-mentioned medium and appropriately selecting conditions under which the cells produce the maximum amount of the target protein and efficiently secrete it in a conventional manner.

 The culturing temperature is usually 15 to 43'C, preferably 20 to 3O'C, and the culturing time is usually about 1 to 1,000 hours. If necessary, it can be cultured under aeration.

 Prior to the main culture, preculture can be performed under the same conditions.

After culturing in this manner, the target protein secreted into the culture supernatant can be collected from the culture by known separation and purification means. For example, the culture is subjected to ultrafiltration or centrifugation to obtain a culture supernatant, and the culture supernatant is subjected to salt precipitation such as ammonium sulfate precipitation, gel-based treatment, treatment with an anion exchanger, cation Purification of the target protein by subjecting it to various treatments such as treatment with an exchanger and treatment with hydrophobic chromatography Can be.

 More specifically, since the target protein of the present invention is easily degraded by a protease derived from a host cell, it is preferable to purify the target protein from the culture by the following method.

 In other words, in the initial stage of purification in which a host cell-derived protease is mixed, a culture solution containing ammonium salt is used in the initial stage of purification by a conventional method such as a centrifugal separation method or an ultrafiltration method. This is a method of treating by column chromatography under the condition of low protein S.

 The buffer used here is not particularly limited as long as it is a buffer containing an ammonium salt. Preferably, it is a buffer having a buffering capacity in a neutral region (pH 6 to 8, preferably about pH 7), and the concentration of the contained ammonium salt is preferably 0.05 to 5 M. Specifically, 0.05 to 1.5 M ammonium phosphate buffer

(Containing (NH <) ₂ HPO * and NH <H ₂ PO 4, pH 6-8). By using a buffer containing an ammonium salt, the present invention can effectively suppress the reduction of the molecular weight of the target protein by a protease, and can purify and obtain an intact protein in a high yield.

 Column chromatography includes ordinary protein purification such as anion exchange column chromatography, cation exchange column chromatography, gel permeation column chromatography, hydrophobic column chromatography, chelate resin column chromatography, etc. Column chromatography used for purification of proteins produced by gene engineering techniques is exemplified. Among them, a preferred embodiment of the present invention includes a combination of hydrophobic force column chromatography and positive ion exchange column chromatography.

As a method for isolating the target protein from the culture supernatant, aqueous column chromatography and cations are preferably carried out using a buffer solution containing ammonium salt, more preferably using an ammonium phosphate buffer solution (pH 6 to 8). There is a method of performing exchange column chromatography. By such a method, the target protein having high specific activity can be isolated at a high yield with a small amount of intact with a small amount of degraded product. The treatment by the hydrophobic chromatography used in the present invention can be performed according to a conventional method. Examples of the hydrophobic chromatography carrier include an insoluble carrier having an alkyl group having 4 to 18 carbon atoms (such as a butyl group, an octyl group, and an octyldecyl group) or a phenyl group. Preferable examples include a fuunil base type, and specific examples include phenyl cellulose (trade name: phenyl cell mouth fine, manufactured by Seikagaku Corporation) and phenyl agarose (trade name: phenyl sepharose, manufactured by Pharmacia). Is exemplified. Adsorption and elution conditions can be appropriately selected according to the target protein to be purified.

 In the case of blow-mouth kinase, the contact conditions include pH 6 to about 8, preferably pH 7 and a salt concentration of about 0.5 to 1.5M, preferably about 0.5 to 0.8M. The elution conditions are about pH 6 to 8, preferably about pH 7, a salt concentration of about 0.05 to 0.7 M, preferably about 0.1 to 0.5 M, and more preferably about 0.2 M.

 The treatment by cation exchange chromatography can also be performed according to a conventional method. As the cation exchanger, any insoluble carrier having an intestinal ion exchange group can be used. ¾Examples of the ion exchange group include a carboxymethyl (CM) -based type and a sulfopropyl (SP) -based type. It is preferably an SP type, and specific examples include SP-agarose (trade name: S-Sepharose, manufactured by Pharmacia), SP-dextran (trade name: SP-Sephadex, manufactured by Pharmacia) and the like. You. Preferred conditions for adsorption and elution can be appropriately selected according to the target protein to be purified.

 In the case of blow mouth kinase, the contact conditions include pH 6 to about 8, preferably pH 7 and salt odor of about 0.01 to 0.2M. The elution conditions include about pH 6 to 8, preferably about pH 7, and a salt concentration of about 1 to 1M.

According to the present invention, a protein that can be reduced in molecular weight by a protease produced by a genetic engineering technique or an enzymatic method can be produced in large form in large size in a complete state. That is, by culturing a host cell that secretes and expresses the protein in the medium referred to in the present invention, it enhances the expression of the host cell, improves the production S of the protein, and secretes the protein from the host cell. Is promoted. Further, according to the present invention, it is possible to effectively suppress the low molecular weight of the protein secreted into the culture medium.

 Among them, the nonionic surfactant used in the present invention does not directly contribute to the growth of host cells or the suppression of protein depolymerization, but has an effect of suppressing the disappearance of proteins by adsorption to instruments and the like. Amino acids, especially arginine and glutamic acid, have the effect of increasing the growth of host cells and inhibiting the production of low-molecular-weight proteins. In addition, ammonium phosphate effectively suppresses the reduction of protein molecular weight. Furthermore, by combining these, the growth of host cells and the production and secretion of proteins are synergistically improved.

 Further, according to the unification method of the present invention, it is possible to effectively suppress the decomposition of proteins that are liable to be reduced in molecular weight at the stage of production, and to efficiently purify and intact intact proteins in high yields. Further, the present invention can provide, for the first time, a Pichia yeast (transformant) capable of secretory production of a blow mouth kinase.

 Examples and experimental examples

 The present invention will be described in more detail with reference to Examples, Experimental Examples and Reference Examples below, which should not be construed as limiting the present invention.

 All enzymes used herein were obtained from commercial sources, such as Takara Shuzo Co., unless otherwise noted. Buffers and reaction conditions for enzyme reactions were according to the manufacturer's recommendations for each enzyme unless otherwise noted.

% Is w / v% unless otherwise specified. NH ₄ -PO * refers to ammonium phosphate buffer containing (H *) ₂ HPC and thigh 4 H ₂ PO4, and Na—P0 * refers to Na ₂ HPO «and NaH ₂ P 0«. Means the contained sodium phosphate 锾 city liquid.

Example 1 Construction of Blow Mouth Kinase Expression Vector, PK0126

After digesting pUC19 with Hindlll, fill-in was performed with Klenow fragment of DNA bolimerase, and ligation was performed by adding pXhol linker [d (pCCTCGAGG)]. Ampicillin, isopropyl-1-D-thio-galactopyranoside (IPTG), 5-promo 4-chloro-1,3-indolyl 8-D-galactoside (X -GAL) -containing plasmid was prepared from a blue-colored colony in an LB medium and named pKO110. Size of the Burasumi de is expected that no deletions without pUC 1 9 so different and Xh o 1 to have a recognition site was confirmed _c Note that a transformant having this Burasumi de It is presumed that the reason for the blue color on LB medium containing IPTG and X-GAL was that the inserted DNA fragment was short and that the insertion frame did not shift due to the insertion.

 Plasmid JUKI (Hiramatsu et al., Gene. 99. 235-), an expression vector for Sacch ronyces cerevisiae containing a gene in which a signal sequence derived from the gene and the Blow Mouth Kinase cDNA are connected in frame. 241. 1991, JP-A-3-240493 and JP-A-4-166088) were partially digested with B1II and then digested with PstI to obtain a part of the signal sequence and A 330 bp fragment containing a portion of the oral kinase cDNA was isolated. The amino acid sequence and the base sequence of the signal sequence derived from the mucor rennin gene contained in the plasmid JUK1 are shown in SEQ ID NO: 1 in the Sequence Listing below. T4 polynucleotide kinase and ATP are added to 5 jiig linkers a and b (Table 1) purified using an OPC column (Abride 'Biosystems Japan) and incubated at 37'C for 30 minutes. To phosphorylate the 5 'end.

 Table 1 Base sequences of synthesized linkers a and b. Linker a: 5 ·-TCGAGATGTTGTTCTCTAA-3 '

 Linker b: 3'-CTACAACAAGAGATTCTAG-5 '

These linkers a and b were mixed and heated at 70 for 20 minutes, and then allowed to cool to room temperature. The annealed linker and 330 bp PstI-Bg1II fragment prepared from Brasmid JUK1 were mixed and ligated, followed by ethanol precipitation. © was performed to recover DNA. The precipitate was dissolved in TEClOmM Tris-HC1 (pH8.0), 1 mM EDTA], mixed with a PstI-Bg1II fragment, ligated, and ethanol precipitated S to recover DNA. . The precipitate was dissolved in TE, and digested with PstI-XhoI to isolate a 36-Obp fragment. This fragment was ligated with a 2.7 kb Pstl-XhoI fragment prepared from pKO110 and then introduced into Escherichia coli for transformation. Brasmid was isolated from 39 clones of ampicillinous colonies. Primary screening by restriction enzyme digestion was carried out to select 6 clones of plasmid, and the nucleotide sequence in the vicinity of the introduced linker was examined with a liquid phase sequencer Quensa-1 (Pharmacia). The sequence was as expected (sequence listing, SEQ ID NO: 2). Two clones were selected and named pKO I13 and pKO114 (3.05 kb) (Fig. 1). Mucor. Contains the gene that connects the signal sequence of rennin and the cDNA of the blow mouth kinase in frame. An 89 Obp PstI-BamHI fragment prepared from plasmid N302Q, an expression vector for Sflccfca mycM cerevisiae, was cloned into pUC19 to obtain pKO111. Further, a PstI—BamHI fragment of 890 bp from pKO111 was prepared, and this fragment was cloned into the PstI—BamHI site of pKO113 to obtain plasmids pKO116 and pKO116. 11.7 (3.95 kb) was obtained (Fig. 2). Brasmid N302Q is aspartic acid, the 302th amino acid from the N-terminus of the brass-mouth kinase cDNA of Brasmid JUKI. Codon AAT of glutamine to CAG of glutamine, site-specific mutagenesis method (Molecular Cloning, A Laboratory Manual. Second edition. Sambrook. J. et al., 1989, modified by Cold Spring Harbor Laboratory, New York), in which the asparagine-linked glycosylation site has disappeared.

pKO116 was digested with BamHI and ΚρηI to separate a 3.95 kb DNA fragment. On the other hand, p SV—G 1 -UK, which is an expression vector for mammalian cells containing blow mouth kinase cDNA (see Japanese Patent Application Laid-Open No. 63-105675 (EP-A-2656784)). A 320 bp DNA fragment was obtained by digestion with BamHI and KpnI, ligated to a PKO116 DNA fragment (3.95 kb), and excised pKO 118 was obtained. ρΚΟ118 was digested with ΚρηI, blunt-ended with Τ4DNΑvolimerase, and then added with pXhoI linker (Takara Shuzo). The 1.57 kb fragment was separated by XhoI digestion and cloned into the XhoI site of pKO110 to obtain the plasmid pK0121. PK0121 was digested with XhoI to isolate a 1.57 kb DNA fragment, which was cloned into the XhoI site of pA0807NX. PKO126 was isolated with the insert inserted in the forward direction (Figures 3 and 4). Ο pAO807Nx is a cloning site for plasmid pHIL-D2 (Invitrogen) Ec This is a plasmid in which the oR I recognition site has been converted to an Xhol recognition site (Esco R I digestion of the plasmid pH IL-D2, fill-in with Klenow fragment, addition of pXho I linker, and then Xho I digested fragments were ligated and ligation reaction was performed to prepare pAO80707NX).

Example 2 Obtaining a transformant

 PKO126 (1Oiig) obtained in Example 1 was digested with zymorylacese, and treated with calcium chloride to make it a competent Pichia yeast iPichia pastoris GTS 115: NRRL deposit number Y—15851) HI S + transformant was selected by introducing into suspension S 100 / z1. The transformant was stroked on a plate containing SD CO. 67% non-containing amino acid YNB, 2% dextrose] to starve a single colony. Thirty clones were isolated as transformants into which pKO126 was introduced, and were named SC01 to SC30.

 The properties of each strain were examined in order to select a strain having a high prokinase production amount from the blow-mouth kinase-secreting strains SC01 to SC30 obtained above.

 (1) Stability of HIS 4 gene

SC 02, 06, 08, 1 0, 1 2, 1 3, 1 8, 1 9, 20, 22, 24, 25, 28 and 14 strains of SC 29 are plated on YPD plates and incubated at 30'C for 2 days Cultured. Inoculate a single colony with 10 clones into HI S + selective plate [Noble agar 15g, sorbitol 91g, glucose 10g, YNB (w / o) 3.35g, biotin 0.2fflg / 500ml], and incubate at 30'C for 2 days Cultured. Proliferation of all colonies was observed. It was also thought that HIS 4 gene stably maintained.

 (2) Culture

The cells were cultured in at 30 ^e C 1 above the 4 strains 3 Om 1 test tube 5 m 1 Y medium (1% methanol-containing YPM medium), 46, 70, 9 of 9 hours after blowing port kinase activity CPA activity (Plasminogen activator activity) as an index], RPHA

(reversed passive haniagglutination), A ". As a result, the PA activity was relatively high in SC06, 08, and 12. A". Are those with an absorbance of 20 or less (SC 0 6, 08, 10, 9, 22) and those with an absorbance of 30 or more (SC 0 2, 12, 2, 3, 18, 19, 24, 25, 28 , 29). Among these strains, SC06 with relatively high PA activity and poor growth rate and SC12 with good growth rate were used in a 50 ml YPMZ 300 ml flask with the methanol concentration changed to 1, 2, 3, 4, 5 And cultured. As a result, in SC06, about 20 IU / m was cultured in a medium containing 2% and 3% methanol S for 71 hours, and in SC12, about 4.6 hours in a medium containing 3% methanol for 71 hours. Expression of 1 UZm1 was observed.

 (3) Wes evening blot

SCO 2, 06, 13 and SC 28 were cultured for 2 days in YPM, medium (YPM medium containing 1% methanol) and YPN! * Medium (YPM medium containing 4% methanol), respectively. A stamp lot of the culture was performed. Band was observed in YP M ₄ medium about 50 kD a in culture cleansed of its bands are bought瘼especially in SC O 6.

 (4) Southern blot

DNA was extracted from SCO 2.06, 10, 12, 19, 22, 29 and Pichia yeast GTS-5, respectively, and the presence of the expression cassette on the chromosome was examined by Southern method. The probe consisted of a 0.9 kb fragment of PHIL-D2 (manufactured by Invitrogen) corresponding to the AOX1 promoter digested with NotI-EcoRI and pMTO15 (corresponding to the structural gene for perkinase). XUCl-digested 1.2 kb fragment of pUC18 into which Blow-mouth kinase cDNA was incorporated) was hybridized with a DNA fragment digested with KpnI, PstI, and NcoI. Isezio Performed. From these results, it was expected that the SC10, 19 and 22 strains, which had poor growth and low PA activity, would be present in a single copy with the expression cassette substituted at the AOX1 site. The degree of proliferation is bad was present in facedown state the PA activity is high SC 0 6 strains expression cassette is replaced with AOX 1 site Double copy _e - way, good SC 02 and 1 2 strains of增殖degree凳 The current cassette was integrated in a form inserted into the AOX1 site in multiple copies, and the SC29 strain was expected to have the expression cassette integrated into a site other than the AOX1 site (Table 2). In the table, S means s 1 ow.

 Table 2

 Existence status of SC strain expression cassette Clones Proliferation PA activity Existence status Existence site

S C 1 0 S + single Replace A0X-1

SC 1 9 S + single Replace A0X-1

S C 22 S + single replacement A0X-1

S C 0 6 S ++++ 2 Replacement A0X-1

SC 02 10 ++ multi import A0X-1

S C 1 2 + Ten + Ten multi Insert A0X-1

SC 29 + + multi insertion

Example 3 Culture of transformant

Blow-mouth kinase secretion-expressing strain SC06 obtained in Examples 1-2 was pre-cultured in YPD medium for 30 days, subcultured twice for 2 days, and 0.01% Triton was added to YPM ₂ medium containing 2% methanol. X- 100, 0.1 M arginine _{hydrochloride, 2% (NH4) 2 HPO} , and 540 nm in _2% NH ₄ H 2 P0 ₄ medium 0.5 liters obtained by blending Absorbance (A _S4.) Is inoculated so that 0.1, and cultured for 3 days in 3 O'C.

Example 4 Blow Mouth Kinase Simplex

 (1) Phenylsilpharose-single column chromatography D-matography

To 2.8 liters of the culture obtained in Example 3, 772 ml of 2M Nt—PO pH 7.0) was added to prepare a final g degree of 0.7M NH * -PC. Equilibration buffer 0.7 M NH * -PO «(pH 7.0) Equilibrated with a Phenyl Sepharose column (5 cm i.d. x 7.8 cm: 150 ml, manufactured by Pharmacia) was added at a flow rate of / hr. After the addition, the mixture was washed with 750 ml of a buffer for equilibration (washl), and further washed with 750 ml of a washing buffer [0.5 M NH «-PO (pH 7.0)] (washll). Thereafter, for elute buffer [elute 〖: 0. 2M NH «-PO (. PH7 0) ] 1 50 m 1 each were collected 8 duty at 1200 m 1, further elution buffer Celute II: 0.05M NH ₄ one P0 (pH 7.0)] 750 ml, elution buffer Celutelll: 1% Triton X—100, 0.05 M Na—PO, (pH 7.0). When the PA activity of each surface was measured, the active components were hardly eluted in the pass eluate and wash I, and 6% for washll, 90% for elute I, 0.4% for elute II and elutelll each, and elute I Most of the activity was observed.

 Next, when each eluted fraction was subjected to a Western blot with an anti-UK antibody, no band was observed in the pass eluate and wash I, and 5 OkDa and 47 kDa were present in the same amount in washll. I was No band was observed in elute II, and most of the degradation products of 30 kDa or less were eluted in elutelU. In elute〗, a clear band of 47 kDa other than the band of 50 kDa was recognized in the facets 1 and 2, and after fraction 3, there was little child of 47 kDa other than the main band of 50 kDa. However, some degradation products of 40 kDa or less were present. Therefore, fraction 3 to fraction 6 which had a low content of 47 kDa and a high PA activity were subjected to the next step.

 (2) S Sepharose column chromatography

1% Triton X—100 1 2 ml and 588 ml of water were added to 600 ml of the boule of fractions 3 to 6 obtained in (1), and the buffer for equilibration [0.01 S-Sepharose column equilibrated with 1 ton X—100, 0.1 M NH ₄ -PO4 (pH 7.0)] [2.5 cm i.d. x 6.1 cm: 30 m Pharmacia Was added at a flow rate of 6 OmlZhr. After addition, wash with 150 ml of equilibration buffer and wash buffer! : Washll: 0. 1 Na C] _{in 0. 05M Na-P0 4 (} . PH7 0), washed with 0.0 1% Triton X- 1 0 0] 1 5 Om 1, elution buffer [0. It was eluted with 1-0.5 M NaC 1 g gradient in 0.05 M Na-PO * (pH 7.0), 0.01 @ triton X—100] 30 Oml. As a result, almost no PA activity was observed in the pass eluate, washl and washll, and PA activity was observed in the elution area of 0.2 to 0.3 M NaCl, and A ₂ ,. And the absorbance of the sample. When silver was stained for each area, no band was observed up to area 48, and a thin band of 52 kDa was present in areas 50 to 56, and the 50 kDa band was observed in area 50 from area 50. Some degradation products were observed in fractions 57 to 62, which are present at minute 68 and have a strong 50 kDa band.

 (3) Concentration

 In step (2), the area that does not contain the 52 kDa protein but has a high content of the 50 kDa protein is bulged, and is subjected to S-condensation using an ultrafiltration membrane (FILTRON STRRED CELLS: Filtron, Technology). did. The PA activity of 1 Oml of the concentrated solution was 43000 IU / m1, and a recovery of 90% was observed. In addition, PA activity of 105 IU / m1 was observed in 52 ml of the pass fraction.

For each step, the amount of protein (modified Lowry method, Bensadoun A. & Weinstein D .: Anal. Biochem., 70, 241-250. 1976), PA activity, specific activity, recovery, and purity were determined (Table 3)

¾3

Bacterial Kinase Protein Content of Blow-mouth Kinase of S C 0 ^ Pla.

 `` Stitching sew au¼)

(ml) C ^ gmD-Ong ) CIU / nil) - (IUxi0 3)

2870 3861108 335961 975 100 1.0

 Ϊ́ / Η: τπ-

 3640 260 · 946 265 · 965 1020 丄 UU 丄 .1

 600 19-11 1120 672 58 947 70 60.5

 S-sef n-s

 1200 9-11 580-696 64 444 72 66.1 i 62 563.5 7700-477 137 500 50 141.0

10 2848 43000, 430 151 408 45 155.0

In the elution stage of Funil Sepharose, protein i was reduced to about 1%, and the purity was increased 60-fold. Furthermore, in the S-Sepharose elution stage, the degree of purification increased to 141 times, and the final concentrate had a PA activity of 4.3 X 10 ^s IU, a protein amount of 2.8 mg, and a specific activity of 1.5 X 1 0 ⁵ I UZmg, recovery 4 5%, degree of purification was 1 55 times.

 (4) Confirmation of the properties of the obtained blow mouth kinase

To confirm the enzymatic properties of the obtained purified properokinase, the Km value was determined and compared with the Km value of natural blow-mouth kinase (hereinafter, referred to as thrombolyse) produced by human Xu cells. As a result, the Km value for the synthetic substrate S-2444 (color-forming synthetic reagent, manufactured by Kabi-Vitrium) was 69 ^ M for thrombolyse, and almost the same as 75 / M for the prokinase of the present invention. Also by Brasmin The change over time under the activity of blow mouth kinase was not significantly different from that of thrombolyse.

 Further, the N-terminal amino acid sequence of the obtained purified blow-mouth kinase was determined. Add 2X BJ (Blue Juice: lO OmM Tris HC 1 (H6.8), 4¾SDS, 0.2% bromophenol blue, 20 glycerol) to the S condensate 301 obtained in (3) 30 / Add 1/1 and heat at 95'C for 5 minutes. Ablate SDS-PAG plate (10/20, Daiichi Kagaku) at 15 / z 1 in 4 lanes. After swimming, PVDF membrane At 7 OmA for 4 nights. The blocking buffer used was 25 mM Tris, 192 mM glycine, and 20% methanol. After blotting, the membrane was washed twice with distilled water for 5 minutes. Coomassie staining solution (0.1% CBB R—250 in 50% methanol) was applied to 100 ml for 10 minutes, and the decolorizing solution (50% Methanol, 10% drunk acid) Decolorized 3 times with 80 ml for 5 minutes, and further washed 6 times with 10 Oml of distilled water for 10 minutes. Finally, it was washed once with 60% methanol and air-dried. The band around 50 kDa was cut out, and the N-terminal amino acid analysis of this peptide was performed. As a result, the N-terminal amino acid sequence

Ser-Asn-Glu-Leu-X-Gln-Val-Pro-Ser-Asn-Cys-Asp-Cys-Leu-Asn-Gly-Gly-Thr Sequence matched. In addition, the expression vector used was a form in which cDNA of blow mouth kinase was ligated to the signal sequence of mucor 'rennin, but it was confirmed that it was processed at a normal position to produce blow mouth kinase. Was done.

Experimental example 1 Effect of each additive in the medium

In order to see the effect of each additive in the culture medium on the growth of the blow-mouth kinase, etc., five kinds of YPM ₂ medium (1% yeast extract, 2% pactopeptone, 2% methanol) were added. Prepared methanol medium

 Medium 1 skin

Medium 2 YP ₂ + 0.01¾ Triton X-100

Medium 3 YPM ₂ + 0.1M arginine hydrochloride

Medium 4 YPM ₂ + 2¾ (NH ₄ ) ₂ HP0 ₄ + 2¾ NH4H2PO4 Medium 5: YPM ₂ + 0.01¾ Triton X-100 + 0.1M Luginin hydrochloride

2¾ (ΝΗ «) ₂ ΗΡ04 + 2¾ Η4Η ₂ Ρ04

The blow-mouth kinase secretion-expressing strain SC06 obtained in Examples 1 and 2 was cultured in YPD medium for 30 days, subcultured twice for 2 days, and placed in a 300-ml 1-volume flask. The medium 1 to 5) was inoculated into 50 ml of a methanol medium so that the absorbance (A _{s <} .

Sampling every 24 hours, A _e *. Measurement, PA activity measurement, and Western plot with anti-UK antibody were performed. Fig. 5 shows the cell growth curve.

 Medium 1 and Medium 2 showed almost the same curve, and A "was changed from 2" to 2 to 23, and then decreased. Medium 3 increased to 41.4 on day 3 and then decreased. The growth on day 1 was very slow in medium 4 and medium 5, but the growth rate then increased, increasing to 36.4 on day 4 in medium 4 and 42.1 in medium 5. Since then, it has decreased.

 FIG. 6 shows the PA activity by the fibrin plate method.

 On day 3, medium 1 showed an upper bound of 71 IU / ml, and then decreased. Medium 4 was lower than medium 2 and about twice as high. In the medium 5 to which all additives were added, the activity was remarkably increased, and was 867 IU / ml on the fifth day.

The culture supernatant from day 2 to day 6 was observed over time using a ゥ stamp lot. On the second day when PA activity appeared in Medium 1, almost no 50 kDa band of complete blown-mouth kinase was recognized, and the 33 kDa and 29 kDa bands were predominant, followed by a decrease in the 33 kDa band. Only the 29 kDa band remained. In the case of medium 2, the tendency was similar to that of medium 1. In medium 3, 50 kDa and 47 kDa bands were present in equal amounts on the second day, the 50 kDa band decreased on the third day, and the 50 kDa and 47 kDa bands disappeared from the fifth day onward. A degradation product of 35 kDa from kDa was added. In Medium 4, the 50 kDa band was predominant until day 3, but then decreased. However, the amount of degradation products from 29 kDa to 35 kDa as in the medium 3 was small. In the medium 5, up to the sixth day, the band of 50 kDa was mainly contained, and the amount of degradation products was small. From these results, it was found that the addition of 0.01 kappa Triton X-100 did not affect bacterial growth or suppress the molecular weight of prokinase, but the activity was increased by more than 3 times. _e which is expected to have significantly suppressed adsorption to the flask mouth kinase

Addition of 0.1 M arginine promoted the growth of the bacterium about twice, and showed a moderate inhibitory effect on low molecular weight. Addition of ammonium phosphate promoted the growth of the bacteria about 1.5 to 2 times, and significantly suppressed the low molecular weight up to the third point. When Triton X-100, arginine and ammonium phosphate were added, a synergistic effect of each additive was observed. Eight activities increased to about 10-fold when cultured in the ^: culture medium, and low molecular weight was also significantly suppressed.

Experimental Example 2 Addition of nonionic surfactant and amino acid to medium

 Blow-mouth kinase secretion-expressing strain SC06 prepared in Examples 1-2 was cultured overnight in YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose) for 30 days. Then, inoculate 30 ml of each of the culture media listed in Table 4 in a 300-ml one-volume flask so that the "" becomes 0.1 degree of S. The culture solution was cultured at 30'C for 72 hours and 144 hours. A, «, and PA activity were measured (Table 4) In the following table, Arg indicates arginine hydrochloride, and Glutamate indicates sodium glutamate.

¾4 Triton X-100 and DfT Luginin Verification HDi ^ g (Jihachihachi * 8, * ₀ ) Medium i2m mm

(Care / A, ") (Care / A _s ")

28/1 3 13/1 7

mi ₂ + 0.01% TritODX-100 130 / 14.5 115 / 11.5

 m + ClMArg 45 / 26.1 12 / 18.4

YPUj + 0.01¾ TritonX-100 +0.1 Arg 310/2 5 380 / 16.8 In the 72-hour culture using the medium containing 0.01% Triton X-130, the degree of growth was the same as that in the basal medium, but the PA activity was 4.6 times that of the basal medium. In the medium supplemented with 0.1 M arginine, the proliferation was enhanced, the PA activity was 1.6 times that of the basal medium, and in the medium supplemented with 0.1% Triton X-100 + 0.1 M arginine, the PA activity was increased in the basal medium. 1 rose 1 times.

 After 44 hours of culture, the PA activity of basal medium, medium supplemented with 0.1% Triton X-1000, and medium supplemented with 0.1 M arginine tended to decrease compared to that after culture for 72 hours. However, it was poor in the medium containing 0.01% Triton X-100 and 0.1M arginine.

 Next, these culture supernatants were subjected to Western blotting using an anti-UK antibody.c. Only a degradation product of 30 kDa or less was observed in the basal medium cultured for 72 hours, and 47 kDa was added when Triton X-100 was added. With the addition of arginine, a light band of 50 kDa was observed. When Triton X-100 and arginine were added, dark bands of 50 kDa and 47 kDa were present.

Experimental Example 3 Effect of adding phosphate and ammonium salt to the culture medium

After culturing SC06 strain in YPD medium overnight at 3 O'C, inoculate 30 ml of each medium described in Table 5 in a 300 ml flask so that A "is 0.1 S degree. The cells were cultured at 30 for 65 hours (Table 5).

Table 5

 Recognition of adding ammonium phosphate

I <Mouth 1 U / till no A n n

Basic medium 5 9 1 3.2

 Medium 1 6 1 1 6.4

 Medium 2 1 28 1 5.6

 Medium 3 1 54 1 7.6

Medium 4 1 76 1 7.9 Basic medium: YP ₂

Medium 1: ΥΡΜ ₂ + 1¾ Η * Η ₂ Ρ0 «+ 1¾ (NHJ ₂ HP0 ₄

Medium 2: YP ₂ tens 0.67¾ NH ₄ H ₂ P0 ₄ Ten _{1.33% (NH 4) 2 HP0} *

Medium 3: YPM ₂ + 2¾ NH4H ₂ P0 «12¾ (NH aHPO *

Medium 4: YPM ₂ + 1.33¾ NH * H ₂ P0 * + 2.67¾ (H ₄ ) ₂ HP0 «

_{1% NH 4 H 2 P0 4} + 1% (NH 4) 2 HPO4 is been made the same as YPM ₂ basal medium and PA activity in supplemented _{medium, 0. 67% NH 4 H 2} PO * + 1. 3 3% (H ₄₎ is a ₂ HP0 ₄ added medium showed activity increase of about 2-fold. _{Further, 2½NH «H 2 PO, +} 2 (NH 4) 2 HP0 4 added medium and _{1. 33% NH <H 2 PO} « +2. 67% (NH 4) 2 HPO * In addition medium of about 3 times the activity A rise was noted. On the other hand, when (NH *) 2 HPO * alone was added, growth was inhibited at both 2% and 4%, and no PA activity was observed.

Experimental Example 4 Effect of adding nonionic surfactant, amino acid, phosphate and ammonium salt to the culture medium

Similarly, add Triton X-100, Arginine, Phosphoric Acid Effect of addition of ammonium salt on cell growth and PA activity was examined <

 Table 6 Triton X-100, abnin, ammonium phosphate (10%) # medium PAS (IU / ml) \ 540 Ground 62 11.9

 Medium 1 110 16.8

 Medium 2 620 22.8

 Medium 3 870 28. 9

: YPM ₂

Medium _{1: ΥΡΜ 2 + 2¾ ΝΗ 2} Ρ0 <+ 2¾

Medium 2: YPM ₂ + 0.01% TritonX-100 + 0.1M Arg

Medium 3: YP ₂ + 2¾ NH4HzP04 + 2¾ (H4) 2HP04 + 0.01¾rritonX-100 + 0.1M Arg

Table 6 shows that when 2% NH ₄ H ₂ PO * + 2% (NH ₄ ) 2 HPO4 was added, the growth was promoted and the PA activity was higher than that of the Triton X-100 + arginine supplemented medium. It also rose 1.4 times. A stamp lot using the anti-UK antibody on this culture was performed. In YPM ₂ medium was not 50 kDa band, but 5 0 kDa band was observed in 4% phosphate ¾ Anmoniumu added. When Triton X-100 arginine was added, there were bands at 50 kDa and 47 kDa, and the band at 47 kDa was deeper, but when 4% ammonium phosphate was added, the band at 50 kDa was predominant. The 47 kDa band was few. From these results, it was considered that the addition of 4% ammonium phosphate suppressed the reduction of the protein from 50 kDa to 47 kDa (Fig. 7).

Experimental Example 5 Glutamic acid, benzamidine, abrotinin, casein Hydrolyzate (casamino acid, NZ-amine, NZ-Case), ammonium salt, and the addition of phosphate

The effects of NH * C1 and Na-PC were investigated to determine whether ammonium phosphate, which was effective in Experimental Example 3, had an effect on phosphate or an ammonium salt. Further, a basic medium culture locations plus 0.0 1% Triton X- 1 00 in YPM _2, it was investigated the effect of增¾ and blanking π Urokina Se 'production of cells by adding the compound.

After culturing SCO 6 overnight in YPD medium, the cells were inoculated into 5 ml 1 Z tubes for each culture so that A ^ n was 0.1 jg, and cultured in 3 O'C for 70 hours. Table 7 shows the results.

Arginine, ben-glutamate; 7-midine, abrotinin, casein hydrolyzate, ammonia

(PA activity Roh A _S ")

IU / ml / A 540

1 YP 2 + 0.01¾ Tri ton X-100 50/12

2 YP ₂ + 0.01% Tri ton X-100 + 0.1M Arg 125 / 23.0

3 YPM ₂ + 0.01% Triton X-100 + 0.1M Glutanate 215 / 25.9

4 Ρ ι + 0.01¾ Triton X-100 + Benzamidine 35 / 13.5

5 mia-H) .01¾ Triton X-lOOlOaM Benzamidine 10 / 9.9

6 YFM2 + 0.01¾ Triton Χ-100 + lOlU / ml Aprotinin 56 / 13.2

7 YPM ₂ + 0.01¾ Tri ton X-100 + 3¾ Casamino acid 410 / 41.8

8 YPMi-K) .01% Tri ton X-10Of3¾ NZ-Amine 105 / 23.9

9 YPMj + 0.01% Tri ton X-10M Z-Case 59 / 19.5

10 ΥΡΜί + ΰ.01¾ Tri ton Χ-100 + ・ thin ^ ΡΟ, + Σ ^ ΟΪ) 2HPO4 79/1 3 11 ΪΡΜ ₂ + 0.01% Triton X-lOOtmCl 11 / 3.6

12 ΪΡΜ2 + 0.01¾ Triton X-100 + 0.25M Na-P0 4 (pH6.0) 2 / 20.4

13 YPM ₂ -K) .01¾ Triton X-100 + 0.25M aP (pi .O) 4 / 17.9

_{14 YPM 2 + 0.01¾ Tri ton X} -100 + 0.25M Na-P0 4 CpiTT.5) 41 / 19.2

30 ^e C70 hrs.5nil / tube culture

The PA activity was increased 4.3 times with the addition of 0.1 IN! Glutamic acid compared to the basal medium, and was more effective than with the addition of 0.1 M arginine. Addition of benzyl-midine at lmM or 1 OmM reduced PA activity. 10 IU / ml abrotinin did not affect PA activity. In addition, 3% casamino acid of the casein hydrolyzate promotes cell growth, An increase in PA activity about 8 times that of this medium was observed. In addition, 4 CNH ₄ C 1 suppressed cell growth and production of blow-mouth kinase. 0. 25M Na-P0 ₄ at pH 7. 0 or less in addition增¾ were subjected to Western plot also by PA activity was low _c anti UK antibody on these culture cleanse regardless're promoted.

 Addition of glutamic acid, benzamidine and abrotinin resulted in a predominantly 50 kDa band and a small 47 kDa band. In 3% casamino acid, 5 O kDa and 47 kDa bands were present to the same extent. At 4¾NH «C1, only a 50 kDa band was observed, and almost no degradation products of 30 kDa or less were observed. With the addition of 0.25 M Na-PO, a 47 kDa light band was observed under the 50 kDa band under the culture condition of pH 7.5.

 From these results, it is recognized that ammonium salt has an effect of remarkably suppressing the reduction of the molecular weight of the 50 kDa protein.

Next, experiments were conducted in which casamino acid, glutamic acid, abrotinin, and benzamidine, which were effective in the above experiment, were added. Table 8 shows the results.

Table 8 Casamino glutamate abrotinin, 'benmidine. Phosphorus si

 ma <D-(? Active eight, ")

IU / ml / A 540

1 YPM2 + 0.01¾ Tri ton X-100 63 / 13.4

2 01¾ Tri ton X-100 + 2¾ Casamino acid 510 / 35.7

3 YP ₂ -K) .01 Triton X-1003 Casamino acid 490 / 42.3

4 YP a + 0.01¾ Triton X-100 + 4¾ Casamino acid 370 / 43.9

5 YPM2 + 0.01¾ Triton X-10 (H5¾ Casamino acid 265 / 45.0

6 YPMz-Kl.01¾ Triton X-100 + 3¾ Casamino acid + 0.1M Glutamte 510 / 43.5

7 YPMz + 0.01¾ Triton X-10 (H3¾ Casamino acidflOIU / ml Aprotinin 560/4 6

8 YP ₂ + 0.01¾ Tri ton X-10 (H3¾ Casamino acid + Benzamidine 470 / 38.7

30 * C, 71 hrs.5ml / tube culture

When the casamino acid concentration was changed from 2% to 5% in 30'C, 7 hour column, PA activity was about 500 IU / m1 at 2-3%, but decreased at 4% or more. The trend was When 0.1 M glutamic acid, 10 IU / ml abrotinin and ImM benzamidine were added to 3% casamino acid, the PA activity was almost the same as that of 3-casamino acid. Western blots were performed with anti-UK antibodies on these cultures. With the addition of 2% to 5% casamino acid, the higher the agricultural level of casamino acid, the less the degradation was, but the bands at 50 kDa and 47 kDa were almost the same. In addition, when glutamic acid, abuchichun or benzamidine is further added to 3% casamino acid, it is not possible to suppress the reduction of the molecular weight from 50 kDa to 47 kDa. From the results of Experimental Examples 1 to 5, Triton X-100, arginine, glutamic acid, and casamino acid are effective for increasing the production amount.To prevent the 50 kDa protein to 47 kDa protein from becoming smaller, Ammonia salts, especially ammonium phosphate, were effective.

Experimental Example 6 Inhibition of Blown Meridase Degradation at Invitro

 The following experiment was performed to see the decomposition inhibitory effect of each additive.

Bikia yeast GTS 1 1 5 strain YPM ₂ medium at 1 4 4 Toki闉, and cultured in 3 0 ^e C, and滤過the culture obtained in the filter of 0. 22 m was cultured on cleansed. To the culture medium 301, added were each of the additives described in Table 9 (total 17.5 D and blow-mouth kinase (2.5 ul) purified from the culture supernatant of the SCO 6 strain according to Example 3). The reaction solution (50ί) was placed in a siliconized conical tube, incubated at 30 for 24 hours, and the degree of decomposition of Blow α-kinase was so-called “stan-blot”. Is shown in Table 9.

 Table 9

1 2 3 4 5 control culture sup.SO 30 30 30 30 30 CYPM2)

0.1¾ Triton X-100 0 5 0 0 5 0

 2Μ Arg. 0 0 2.5 0 2.5 0

20% intestine ₂ Ρ0 ₄ 0 0 0 5 5 0

Η ₂ 0 17.5 1Z5 15 7.5 0 17.5

 ProU 2.5 5 5 5 2.5 2.5

50 50 50 50 50 50 ul As a result, with the addition of 0.01% Triton X-100, the degree of decomposition was almost the same as with no additive. When 0.1M arginine was added, the amount of degraded products of 35 kDa or less was small, but the amount of degraded products of 47 kDa was slightly observed. Addition of ammonium phosphate, or Triton X-100, arginine and ammonium phosphate, significantly inhibited degradation.

Experimental example 7 N-terminal amino acid sequence of degradation product

When cultured in YPM ₂ medium additives without addition of SC O 6 strain Burouroki kinase full body not already almost present in second day was observed PA activity in a short time 3 O k Da protein (non-reducing conditions ). In order to investigate the structure of this degradation product, the following experiment was performed.

SC 0 6 strain YPM ₂ medium 50m 1 (30 0 m 1 flask) was inoculated, 30 'and cultured for 5 days at C, and the culture liquid 30 0 m 1 1 M Tris-HCl (pH 7.5) Add 16 ml, 1% Triton X-100 3.2 ml, adjust the pH to 7.5 with hydrochloric acid, and apply to an anti-perokinase antibody formyl-cell mouth fine column (manufactured by Nippon Chitz) to equilibrate the buffer. After washing with the solution [5 OmM Tris-HCl (PH7.5), 0.01% Triton X—100], the buffer [5 OmM Tris-HCl (pH7.5), 0.5M NaC0 0.1 Triton X-100] and eluted with an eluent [0.2M glycine hydrochloride (pH 2.5), 0.5M NaCl, 0.01% Triton X-I 00]. . The eluate with PA activity was concentrated approximately 100-fold with molcut L (fraction molecular weight 10,000, UFP2LGC24, manufactured by Miriboa) and Ultra-free C3-LGC (fraction molecular weight 10,000, manufactured by Millipore). The band of 30 kDa was cut out by -PAGE, and the amino acid sequence of the 30 kDa protein obtained by extracting from the band was determined.

 As a result, two peaks having the following amino acid sequence at the N-terminus were observed.

① Ile-Ile-Gly-Gly-Glu-Phe-Thr-Thr-Ile-Glu-Asn-Gln-

② Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Leu-Lys-Phe-

The amino acid sequence of ① matches the sequence from the isoleucine at the 159th position from the N-terminal of the blow-mouth kinase, and the amino acid sequence of ② is 1336 from the N-terminal of the blow-mouth kinase. It matched the sequence from the lysine of the eye. This suggests that the 30 kDa protein is a small double-stranded perovin kinase.

Reference example Measurement of PA activity

 PA activity was measured according to the method of Levin. E. G .. et al. (Fibrin plate method, J. Cell Bio., 94, 631-636. 1982).

After adding 50 parts of 100 U / m1 human thrombin to 1% agar solution of 6 O'C, 50 mM sodium phosphate buffer (PH7.0), 100 mM NaCl, 0. the solution of 37'C which was dissolved 1 ¾ Ushifuiburino one Gen in 1 ¾NaN ₃ were mixed in equal amounts was added to a horizontal plate, to solidify the agar. Drill a hole of 4 mm in diameter in the solidified cloudy agar layer, add 20 μl of sample to each hole, and leave at 37'C for 12 hours. In the presence of PA activity, the transparent agar layer becomes transparent with a dissolved circle. The diameter of the lysis circle is measured, and the PA activity is measured by comparison with a standard Blow-mouth kinase.

Sequence listing

SEQ ID NO: 1

Array length: 5 4

Sequence type: nucleic acid

Number of chains: single strand

 Topology: Direct desire

Sequence type: genomic DNA

mm

 Brasmid J UK 1

Array features

 1-54 S sig peptide

Array

 ATG TTG TTC TCT AAG ATC TCC TCT GAT ATC TTG TTG ACC GCT GCA AGC 48 Met Leu Phe Ser Lys lie Ser Ser Ala lie Leu Leu Thr Ala Ala Ser

 5 10 15

 TTC GCG 54 Phe Ala

 18 SEQ ID NO: 2

^ Column length: 7 2

Sequence type: nucleic acid

Number of towns: one town

 Topology: direct letter

 Column type:

Origin

 PK0113 or ρΚΟΙ

Array features 7-60 E sig peptide

 61-72 E peptide

Array

 CTCGAG ATG TTG TTC TCT AAG ATC TCC TCT GCA ATC TTG TTG ACC GCT 48 Met Leu Phe Ser Lys lie Ser Ser Ala He Leu Leu Thr Ala

 -15 -10 -5

GCA AGC TTC GCG AGC AAT GAG CTC 72 Ala Ser Phe Ala Ser Asn Glu Leu

 1

Claims

The scope of the claims

 1. Host cells that secrete and produce proteins that can be degraded by host cell-derived protease are selected from the group consisting of acidic amino acids, basic amino acids, phosphates, ammonium salts, and nonionic surfactants A method for producing a protein that can be reduced in molecular weight by a host cell-derived porcine protease, comprising culturing the culture in a medium containing at least one of the above-mentioned cultures and collecting the protein from the resulting culture.

 2. The method according to claim 1, wherein the amino acid is at least one selected from the group consisting of arginine, lysine, glutamic acid and aspartic acid.

 3. The nonionic surfactant according to claim 1, wherein the nonionic surfactant is at least one selected from the group consisting of polyoxyethylene mono-p-tert-octylfurunyl ether and polyoxetylene sorbin fatty acid ester. The described method.

 4. The nonionic surfactant is polyoxyethylene (9 to 10) mono p-tert-octyl phenyl ether, boroxy ethylene (9) mono ρ-tert-octyl phenyl ether, 4. The method according to claim 3, which is at least one selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate and polyoxyethylene (20) sorbitan monooleate.

 5. The method according to any one of claims 1 to 4, wherein the host cell is Pichia yeast.

 6. Proteins that can be degraded by host cell-derived proteinases include blourokinase, human tissue plasminogen activator, blood coagulation factor IX, blood coagulation factor X, and protein (:, interferon, evida). The method according to any one of claims 1 to 5, wherein the method is selected from the group consisting of one malgrowth factor, β-endolphin, and a mutant or derivative thereof.

 7. The medium is (a) at least one selected from the group consisting of acidic amino acids and basic amino acids;) at least one selected from the group consisting of phosphates and ammonium salts; and (c) nonionic The method according to claim 1, further comprising a surfactant.

8. The method according to any one of claims 1 to 7, wherein the method of collecting the protein from the culture supernatant comprises treating the culture supernatant by column chromatography using a buffer solution containing ammonium salt. The described method.

9. Column chromatography requires at least hydrophobic column chromatography! 9. The method according to claim 8, comprising a treatment with Zion exchange force ram chromatography.

 10. Pichia yeast secreting and producing blow-mouth kinase is cultured in a medium containing arginine, ammonium phosphate and polyoxyethylene (9-10) mono-p-tert-butyl octylphenyl ether, and the resulting culture is obtained. A method for producing blow-mouth kinase, comprising collecting brourokinase from a supernatant.

 11. The method according to claim 10, wherein the method for collecting blow-mouth kinase from the culture supernatant comprises treating the culture supernatant by column chromatography using a buffer solution containing ammonium salt. Method.

 12. The method according to claim 11, wherein the treatment by column chromatography includes at least treatment by aqueous column chromatography and treatment by cation exchange column chromatography.

 1 3. Pichia yeast secreting and producing blow mouth kinase.