JP2971894B2 - Method for producing C-kinase using yeast - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for expressing in a yeast a protein kinase useful as a reagent or the like (hereinafter sometimes abbreviated as “C-kinase” or “PKC”). Furthermore, the present invention relates to a C-kinase expression vector and a yeast transformed with the vector.

2. Description of the Related Art Various biologically active substances such as cell growth factors, hormones and neurotransmitters are known to be involved in various functions and adaptation phenomena of living organisms. These extracellular signals are exerted through intracellular mediators such as cyclic AMP, cyclic GMP, diacylglycerol (DG), and calcium. In particular, DG has been found to be the result of the degradation of cell membrane inositol phospholipids by many hormones and neurotransmitters that activate cell functions. It is thought that this DG activates C-kinase in the presence of calcium, and the phosphorylation of various intracellular proteins by this oxygen leads to activation of various cell functions and cell proliferation. That is, C-kinase is an enzyme that plays an important role in one of the major signal transduction mechanisms of external signals. The enzyme purified from rat cerebrum is an acidic protein having an isoelectric point of pH 5.6 and a molecular weight of about 77,000.
In addition, this enzyme is made of a single peptide consisting of a hydrophobic part and a hydrophilic part, binds to the cell membrane via the hydrophobic part, and has an active center in the hydrophilic part. C-kinase is usually in an inactive form and is activated by calcium and phosphatidylserine and DG. The phosphate donor is known to be ATP.

As described above, C-kinase is an enzyme which is indispensable for studying the transduction mechanism of extracellular signals, since it also transmits intracellular signals through phosphorylation of proteins as one of protein kinases. , Its value as a reagent is very high. In addition, this enzyme is directly activated by phorbol ester which is a carcinogenic promoter. Phorbol esters not only promote the carcinogenesis process, but also induce cell division and differentiation, induce enzymes,
It is known that it is deeply involved in many biological reactions such as lipid metabolism. These facts indicate that C-kinase is likely to be an important indicator enzyme in various pathological cells and cancer cells caused by abnormalities in the cell membrane receptor transduction mechanism.
It is conceivable that an antibody against C-kinase is used as a diagnostic or test agent. A sufficient supply of C-kinase is required for these purposes. Mass production of animal-derived C-kinases, from which materials are relatively easy to obtain, is also very difficult. Rat C-kinase has already been purified from rat brain, in which case several subtypes have already been successfully cloned [Y. Ono et al., Science.
e), 236, 1116 (1987)], although the protein is also produced by genetic recombination technology, but since it is expressed in animal cells, the production amount is small, and a method for obtaining a larger amount of C-kinase is desired. ing.

Problems to be Solved by the Invention As described above, there are many unclear points regarding the properties of human C-kinase and rat C-kinase. Accordingly, there has been a demand for a method for producing the protein as a reagent or a test agent by genetic recombination technology.

Means for Solving the Problems The present invention relates to (1) Saccharomyces cerevisiae
romyces cerevisiae) NA74-3A (ρ ⁻ ) / pTFE755 (FERM
BP-1976) or Saccharomyces cerevisiae (Sacchar
omyces cerevisiae) NA74-3A (ρ ⁻ ) / pTFE756 (FERM
BP-1977), which contains a DNA encoding the protein kinase C protein and is capable of autonomously replicating in yeast (pTFE755 or pTFE755).
TFE756), (2) a yeast carrying the expression plasmid according to (1), (3) a yeast according to (2), wherein the yeast is a strain deficient in respiratory function, and (4) a yeast, which is Saccharomyces cerevisiae. (2) or the yeast according to (3), (5) Saccharomyce (Saccharomyce)
cerevisiae) NA74-3A (ρ ⁻ ) / pTFE755 (FERM BP-1
976) The yeast according to (2) above, (6) Saccharomyces cerevisiae
The yeast according to the above (2) and the yeast according to any one of the above (2) to (6) represented by NA74-3A (ρ ⁻ ) / pTFE756 (FERM BP-1977) are cultured in a medium. And producing and accumulating protein kinase C protein in the cells.

The vector (eg, plasmid) used in the present invention may be any vector as long as it can be replicated in yeast.
pSH19 [S. Harashima et al., Molecular and Cellular Biology (Mol. Cell. Biol.). 4, 771 (198
4)], pSH19-1 (European Patent Application Publication EP-A-02)
By inserting a promoter into these, a beagle for exogenous gene expression can be obtained.

The promoter used for expression may be any promoter as long as it can function in yeast.
D (GAPH) promoter, PHO5 promoter, PGK promoter, ADH promoter, PHO81 promoter, GAL10
Promoters and the like are preferably used.

A promoter can be prepared yeast from the corresponding gene. Also, it can be chemically synthesized.

The plasmid for expressing the DNA encoding the polypeptide C kinase is a DNA encoding the C kinase polypeptide downstream of the promoter of the above expression vector or the yeast which is located at the 5 'end of the DNA encoding the C kinase polypeptide. This can be obtained by inserting DNA to which DNA encoding all or a part of the signal peptide to be bound is bound. As an expression vector, for example,
pTFE112, pPHO17, pGLD906-1 [Y. Itoh et al., Biochemical Biophysical Research Communication (Biochem. Biophys. Res. Commun.). 138, 268 (198
6)]. In this case, the expression level of the DNA can be increased by inserting a terminator downstream of the DNA encoding C kinase. Examples of the terminator include terminators such as phosphoglycerate kinase gene (PGK), FLP gene of 2 μDNA, and invertase gene (SUC2).

The polypeptide C kinase in the present invention includes human, bovine, rat or rabbit.
For example, Science, Vol. 236, 1116-1120
(1987), page 1117, Table 1, C-kinase.

An expression vector containing a DNA having a nucleotide sequence encoding a polypeptide of C kinase protein in the method of the present invention includes, for example, (a) isolating RNA encoding C kinase; and (b) separating single-stranded RNA from the RNA. (C) integrating the complementary DNA into a plasmid, (d) transforming the resulting recombinant plasmid into a host, and (e) transforming the resulting plasmid. After culturing the cells, the desired DN is obtained from the transformant by an appropriate method, for example, colony hybridization using rat cDNA as a probe.
A. A plasmid containing A can be produced by isolating the plasmid containing A, cutting out the cloned DNA of interest from the plasmid, and ligating the cloned DNA downstream of the promoter in the vehicle. RNAs encoding kinases can be obtained from various C-kinase producing cells, such as brain-derived cells or fibroblasts. For example, W138 is a human fibroblast.
(ATCC number CCL-75) or IMR90 (ATCC number CCL-18)
6). The cells WI38 or IMR90,
The American Type Culture Collection, Luminous Catalog of Cell Lines & Hybridomas (5th editi)
on, 1985.

As a method for preparing RNA from C-kinase-producing cells, a guanidine thiocyanate method [(JMChirgwin
Et al., Biochemistry, 18 , 5294 (1
979)].

Using the RNA thus obtained as a template and reverse transcriptase, for example, the method of H. Okayama et al.
And Cellular Biology (Molecular and Cell
ular Biology) 2, 161 (1982 ) and ibid 3, 280 (198
3)], and synthesize the obtained cDNA into a plasmid.

Methods for constructing expression plasmids in the present invention are known and are described in the literature, for example, Molecular Cloning (1982), Cold Spring Harbor Lab.
oratory).

Examples of a host transformed with the expression plasmid include yeast, and among them, Saccharomyces cerevisiae is preferable. Especially S.Cere
A visiae respiratory deficient strain (ρ ⁻ ) is suitable as a host.

From respiratory yeast (parent strain ρ ⁺ ) to respiratory deficient strain (ρ
^- ) Is known per se, for example [[Laboratory Course Manual for Methods in
East Genetics [Laboratory Course Manual
for Methods in Yeast Genetics, "Cold Spring Harbor Laboratory
Laboratory, (1986)]. That is, after culturing the parent strain in a medium containing ethidium bromide, a respiratory-deficient strain can be easily obtained by isolating a strain that can grow on a medium containing glucose as a carbon source but cannot grow on a medium containing glycerol. In addition, a respiratory-deficient strain can be obtained from a single colony of the parent strain, albeit at a low frequency. When the parent strain referred to herein is a transformant (recombinant) having an expression plasmid, a recombinant having a high gene expression level can be directly obtained by obtaining the respiratory-deficient strain. When the parent strain does not carry the expression plasmid, the respiratory-deficient strain is obtained, and the desired recombinant is obtained by introducing the expression plasmid into the strain.

A method for transforming yeast using a recombinant DNA (plasmid) is a method known per se, for example, the lithium method [H.
Et al., “Journal of Bacteriology (J. Bact.
eriol.), 153 , 163 (1983), protoplast method [A.
nen et al., Proc. of the National Academy of Sciences (Proc. Natl. Acad. Sci. US
A), 75 , 1927 (1978)].

The transformant (recombinant) thus obtained is cultured by a method known per se.

As the medium, for example, Burkholder (Burkholder minimal medium [American Journal of Botany (Amer. J. Bat.), 30 , 206 (1943)] or a modified medium thereof [A. Toh-e et al., Journal of Bacteriology (J. Bacteriol), 113 , 727 (1973)], or a low-phosphate medium [A. Toh-e et al., Journal of Bacteriol (J. Bacteriol), 113 , 727 (1973)]. Usually 15 ° C to 40 ° C, preferably 24 ° C to 37 ° C
C. for 10 to 168 hours, preferably 72 to 144 hours. Shaking culture or static culture may be used, but if necessary, aeration and stirring may be added.

The C-kinase generated and accumulated in this manner can be obtained by a conventional protein extraction / purification method, for example, cell disruption by glass beads, centrifugation, salting out, isoelectric point precipitation, gel filtration, ion exchange chromatography, high-performance liquid Purification can be easily performed by appropriately combining purification steps such as chromatography, affinity chromatography, sucrose gradient ultracentrifugation, and cesium chloride gradient ultracentrifugation.

Action The PKC (protein kinase-C) polypeptide obtained in the present invention has the same enzymatic activity as the PCK polypeptide prepared from rat brain cells, and can be used as a reagent or as a material for a diagnostic kit.

The C-kinase produced here can be used as a reagent or an antibody prepared and used together with the antibody as a diagnostic / test agent. For example, the C-kinase of the present invention can be used as a research reagent for cell membrane receptive transduction, a diagnostic / testing agent for diseases caused by abnormal cell receptive transduction (eg, tumor), or a prophylactic / therapeutic agent for these diseases. Can be used as a screening reagent. For example, 0.001 to 10 μg of C-kinase is used for one diagnosis / test.

In the specification and the drawings of the present application, when bases and amino acids are indicated by abbreviations, IUPAC-IUB Commiis
This is based on the abbreviation by sion on Biochemical Nomenclature or the abbreviation commonly used in the art. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.

DNA Deoxyribonucleic acid RTA Ribonucleic acid mRNA Messenger RNA A Adenine T Thymine G Guanine C Cytonin dATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate dGTP Deoxyguanosine triphosphate dCTP Deoxycytidine triphosphate ATP Adenosine triphosphate EDTA Ethylenediaminetetraacetic acid SDS sodium dodecyl sulfate DTT dithiothreitol Gly glycine (G) Ala alanine (A) Val valine (V) Leu leucine (L) Ile isoleucine (I) Ser serine (S) Thr threonine (T) Cys cysteine (C) 1 / 2Cys Half Cystine Met Methionine (M) Glu Glutamic acid (E) Asp Aspartic acid (D) Lys Lysine (K) Arg Arginine (R) His Histidine (H) Phe Phenylalanine (F) Tyr Tyrosine (Y) Trp Tryptophan (W) Pro Proline (P) Ash Sparagine (N) Gln Glutamine (Q) Ap Ampicillin resistance gene Tc Tetracycline resistance gene ARS 1 Autonomous replication sequence 1 Examples The present invention is described in more detail below with reference examples and examples. Is not limited to this.

The respiratory-deficient strain Saccharomyces obtained in Reference Example 1 was used.
cerevisiae NA74-3A (ρ ⁻ ) was released on October 23, 1987 by IFO
Was deposited as accession number IFO 10431, and the transformant was deposited on FRI on November 5, 1987 as accession number FERM P-9962, and the deposit was switched to a deposit based on the Budapest Treaty, and the accession number FERM was deposited. Stored at FRI as BP-1948.

Further, a transformant Saccharomyces cerevisiae NA74-3A / pT carrying the plasmid pTFL710T obtained in Reference Example 5
FL710T was granted to IFO on October 23, 1987 with accession number IFO 1043
2 and this transformant was obtained on November 5, 1987.
Deposited with the FRI on the day as accession number FERM P-9969. This deposit has been converted to a deposit under the Budapest Treaty and is deposited with FRI under the accession number FERM BP-2090.

Further, the transformant Saccharomyces obtained in Example 4 was used.
cerevisiae NA74-3A / pTFE755 and Saccharomyces cer
evisiae NA74-3A / pTFE756 was deposited with the IFO on July 14, 1988 under the accession numbers IFO 10442 and IFO 10443, and these transformants were deposited on the FRI on July 23, 1988, with the accession number FERM BP-1976. , FERM BP-1977.

Reference Example 1. Preparation of respiratory-deficient strains Laboratory Course Manual for Methods in East Genetics (Laboratory Cou
rse Manual for Methods in Yeast Genetics] [Cold Spring Harbor Laboratory (Cold Sprin)
g Harbor Laboratory), 1986)), using Saccharomyces cerevisiae NA74-3A using ethidium bromide.
Saccharomyces cerevisiae NA74-
3A (ρ ⁻ ) (IFO 10431, FERM BP-1948) was isolated.

Reference Example 2 Preparation of Expression Vector The expression vector pTFE112 was prepared by using the BamHI-XhoI site [GLD of the expression vector pGFE213 obtained in Reference Example 6 described below.
790 bp BamHI-XhoI obtained from pTFL710T obtained in Reference Example 5 described later.
It was prepared by inserting a DNA fragment [GAL10 promoter (shown as GAL10p)] in the forward direction (FIG. 1).

Reference Example 3 Construction of human lysozyme secretion plasmid pGEL735 To 5 μg of E. coli vector pBR322, 1.5 units of restriction yeast BalI were added, and 40 μl of a reaction solution [10 mM Tris-HCl (pH7.
5), 10mM MgCl ₂ , 1mM dithiothreit
ol)] at 37 ° C. for 5 hours, followed by phenol extraction as usual, followed by ethanol precipitation. Xho I linker d [pCCTCGAGG] phosphorylated on this DNA (New Englan
d Biolads) (50 ng), and both were ligated with T4 DNA ligase according to a conventional method.

Escherichia coli DH1 strain was transformed with this reaction solution, and the resulting ampicillin-resistant and tetracycline-resistant colonies were subjected to an alkali extraction method [Barenboim and Biry
m, HC & Doly, J), "Nucleic Asshizu Research (Nucl. Acids Res.)" 7, 1513 (1979)] extracted plasmid was Bal I site to obtain a plasmid pBR322X converted to Xhol I site.

A report by Ikehara et al., “Chemical and Pharmaceutical Bulletin (Chem.Pharm.Bul)
l.) ", 34 , 2202 (1986)], the human lysozyme gene is prepared by dividing it into 52 oligonucleotide blocks as shown in Table 1, for example.

CGAGAGATGCGAAT as U2-taq instead of U2 in Table 1
TCTGGCTAACGCATCTCT as L2-taq instead of L2,
It was synthesized according to the report of Ikehara et al. Next, U2-taq, U3 ~
Oligonucleotide block hybrids were formed using the U26, L2-taq, and L3-L26 fragments according to the report of Ikehara et al. Each of these groups is described in JP-A-61-1587.
No. 93 (European Patent Application Publication No. 181,634) After ligation by the method described in Example 2, both 5 'ends were enzymatically phosphorylated to obtain a human lidozyme gene.

2.6 units of pHBR322X plasmid with 6 units of control enzyme
Xho I and 6 units of the control enzyme Cla I were reacted in a 35 μl reaction solution [33 mM acetate buffer pH 7.9, 66 mM potassium acetat
e, 10mMmagnesium acetate, 0.5mM dithiothreitol, 0.01
% BSA] at 37 ° C for 1 hour, followed by deproteinization with phenol and precipitation with cold ethanol. The DNA (200ng)
Human lysozyme gene fragment prepared as described above
Mix with 100 ng, and add 10μ reaction solution [66 mM Tris-HCl (pH
7.6), 10mM ATP, 10mM spermidine , 100mM MgCl 2, 150mM D
TT, 2 mg / ml BSA, 5 units of T4TNA ligase] at 14 ° C. overnight to bind DNA. Using this reaction solution, Escherichia coli DH1 was transformed by the method of Cohen et al. [Cohen,
SNet al. "Proceding of the National
Academy of Sciences (Proc.Natl.Acad.S
ci.USA) " 69 , 2110 (1972)]. The resulting transformants are further extracted with alkali (as described above).
The plasmid was isolated by PCR, and the molecular weight and the degradation pattern by restriction enzymes were examined. Thus, pLYS221 into which the human lysozyme gene fragment was inserted was obtained. The EcoRI-XhoI fragment of pLYS221 was separated and its nucleotide sequence was determined by the dioxynucleotide synthesis chain termination method. As a result, the expected human lysozyme gene Taq I-XhoI fragment was obtained.

This sequence corresponds to 4 of the amino acid sequence of human lysozyme.
Code from Glu to 130th Val.

Known amino acid sequence of the signal peptide of egg white lysozyme [Jung, A. et al., "Progressing of the National Academy of Sciences (Proc. Natl. Acad. Sci.)" 77 , 5759 (1980) ]
For the purpose of substituting the fourth leucine with phenylalanine, the sixth isoleucine with leucine, and the eighth valine with alanine, the codons with higher codon usage in yeast are given priority for higher expression. To choose. Yeast PGK upstream of ATG to increase expression
Hybrid signal (hybr
The nucleotide sequence was determined in view of allowing for the construction of an id signal. The synthesized nucleotide sequence is shown in FIG. 2 and has an Xho I site at the 5 'end and a Taq I site containing the coding region of human lysozyme at the 3' end. The entire sequence consists of eight oligonucleotide blocks (# 1 to # 8), which are determined by the phosphoamidite method [Caruthers, MH et al; "Tetrahedron Letters" 22 , 1859 (1981)].

First, each of # 2 to # 7 was mixed at 10 μ (5 μg), and 10-fold concentration of Kinase buffer (Kinase b) was added thereto.
buffer) [0.5 M Tris-HCl, 0.1 M MgCl ₂ , 0.1 M mercaptoethanol, pH 7.6] 20 μ, 10 mM ATP 20 μ, T4 polynucleotide kinase (Takara Shuzo Co., Ltd.) 20 μ (50 u), and distilled water 80 μ After reacting for 2 hours at 65 ° C,
The reaction was stopped after treatment for 20 minutes. # 1 and #
8 and 10 μg (5 μg) of each were added, and T4 ligase (NE
(Manufactured by B company) was added thereto and reacted at 10 ° C. overnight. Reaction liquid 1
The gel was subjected to 0% polyacrylamide electrophoresis, and a 76 bp fragment was cut out and extracted from the gel by electrophoretic elution. These were dissolved in 45 μl of distilled water, and added to a 10-fold concentration of a kinase buffer (described above) 6 μm, 10 mM ATP 6 μm, T4
2 µ (5 u) of polynucleotide kinase (described above) was added, and the mixture was allowed to react at 37 ° C for 1 hour and then stored at -20 ° C.

In FIG. 2, amino acids are represented by one-letter symbols (IUPAC-IUB biochemical naming committee confirmation rules).

Example A: Alanine B: Aspartic acid or asparagine C: Cysteine D: Aspartic acid E: Glutamic acid F: Phenylalanine G: Glycine H: Histidine I: Isoleucine K: Lysine L: Leucine M: Methionine N: Asparagine P: Proline Q: Glitamine R: arginine S: serine T: threonine V: valine W: tryptophan Y: tyrosine Z: glutamic acid or glutamine X: unknown or other amino acid 236 μg of the plasmid pLYS221 obtained above was EcoRI (Nippon Gene) 120u, XhoI ( (Nippon Gene)
The mixture was treated with 120u at 37 ° C for 2 hours to cut out the human lysozyme coding region. This fragment was further treated with 26u of Taq I (manufactured by Nippon Gene) at 65 ° C for 1 hour to cut out a human lysozyme coding region partially lacking the N-terminus.

About 1 μg of this fragment was mixed with 0.5 μg of DNA encoding the signal sequence described above, and T4-ligase (supra)
After reaction at 16 ° C for 16 hours in the presence of 0u, XhoI (42
u) processed.

10 ng of the obtained XhoI fragment and the yeast expression vector pGLD906-1 (Japanese Patent Application Laid-Open No. 61-43991, published in Europe)
No. 171,908) was mixed with 1 ng of the vector treated with Xho I, and both were ligated in the presence of T4 ligase.

Escherichia coli DH1 was transformed with this reaction solution by the above-mentioned method, and a number of plasmids having a signal sequence coding region and a human lysozyme gene inserted in the same direction as the promoter downstream of the GLD promoter were obtained. . One of them was named pGFL735 and used in the following experiments.

Reference Example 4 Construction of human lysozyme secretion plasmid pPFL725T A 0.28 kb Aha III-Sal I fragment containing a PGK terminator was isolated from HBsAg-modified P31 expression plasmid pGLDP31-RcT (European Patent Application Publication No. 0235430), and Sal
After ligation of the I linker pGGTCGACC with T4 DNA ligase, Sa
Treated with lI. 0.28k by agarose gel electrophoresis
b Separation of the Sal I fragment from the Sal I linker not bound to the fragment and isolation of DNA using DEAE cellulose paper [Winberg, G. & Hammarskjold, M.-L .), "Nucl. Acids Res., 8 ,
253 (1980)] from agarose gel to 0.28 kb Sal
I fragment was obtained.

Human lysozyme secretion plasmid pGEL125 using egg white lysozyme signal peptide [Yoshimura, K. et.
al.), “Biochemical and Biophysical Research Communication (Biochm. Biophys.
s.Commun.), 145 , 712 (1987), FERM BP-1345]
Cleavage with amHI and XhoI, 1.5 kb BamHI-XhoI fragment containing the GLD promoter and egg white lysozyme signal peptide-human lysozyme coding region and the rest, 8.4 kb
The BamHI-XhoI fragments were separated by agarose gel electrophoresis and each was isolated.

At the 3 'end (XhoI portion) of the 1.5 kb BamHI-XhoI fragment containing the above-mentioned GLP promoter, egg white lysozyme signal peptide coding region and human lysozyme coding region
The 0.28 kb Sal I fragment containing the PGK terminator was ligated with T4 DNA ligase and subjected to agarose gel electrophoresis,
A 1.8 kb BamHI-SalI fragment was isolated.

The obtained 1.8 kb BamH I-Sal I fragment and 8.4 kb BamH I-X
The ho I fragment was ligated with T4 DNA ligase and used to transform Escherichia coli DH1. A plasmid was prepared from the ampicillin-resistant transformant and named pGEL125T.

Human lysozyme secretion plasmid pG obtained in Reference Example 3
A 0.5 kb Xho I fragment encoding the improved signal peptide and human lysozyme was isolated from FL735. Meanwhile, P
Expression vector pPHO17-1 having HO-5 promoter
(European Application Publication No. 0235430) was digested with XhoI, ligated with a 0.5 kb XpoI fragment using T4 DNA ligase, and transformed into E. coli DH1. A plasmid was prepared from the obtained ampicillin-resistant transformant and named pPFL725.

Plasmid pGEL125T obtained as described above and
Both pPFL725 have an XbaI cleavage site upstream of the promoter and in the human lysozyme coding region. Therefore, both plasmids were cut with XbaI and the 1.7 kb Xba
After ligation of the I fragment and the 7.9 kb Xba I fragment of pGEL125T with RT4 DNA ligase, Escherichia coli (E. coli) DH1 was transformed. A plasmid was isolated from the obtained ampicillin-resistant transformant and named pPFL725T.

Reference Example 5 Construction of human lysozyme secretion plasmid pPTFL710T Human lysozyme secretion plasmid pG obtained in Reference Example 3
A 2.3 kb XbaI fragment was isolated from FL735 and a 7.9 kb XbaI fragment was isolated from the plasmid pGFL125T obtained in Reference Example 2, and both were ligated with T4 DNA ligase to transform E. coli DH1. A plasmid was isolated from an ampicillin-resistant transformant and named pGFL735T.

G of plasmid pBM150 [“Molecular Cellular Biology”, 4 , 1440 (1984)]
It was obtained by replacing the 275 bp Sal I-BamH fragment upstream of the al10 promoter with a multilinker (FIG. 3) (about 40 bp) having restriction enzyme sites such as SacII, BamHI, SalI, and BglII. After ligating the EcoRI-XhoI adapter with a T4 DNA ligase to a 0.7 kb BglII-EcoRI fragment containing the promoter from plasmid p286 to Gal10, BglII and XhoI
Treated with I, 0.7 kb Bgl by agarose gel electrophoresis
The II-Xho I fragment was isolated.

On the other hand, contains the GLD promoter of plasmid pGFL735T
The 1.1 kb BamHI-XhoI fragment was removed, and a BglII-XhoI fragment containing the Gal10 promoter was ligated to transform Escherichia coli (DH) DH1. A plasmid was isolated from the obtained ampicillin-resistant transformant and named pTFL10T. Saccharomyces cerevis
iae NA74-3A was transformed with this plasmid pTFL710T and transformed into Saccharomyces cerevisiae NA74-3A / pTFL710T.
(IFO10432, FERM BP-2090).

REFERENCE EXAMPLE 6 Construction of Human EGF Secretion Plasmid pGFE213 In Example 3, # 9 shown in FIG. 4 was replaced with # 9 shown in FIG. 2, and # 10 was replaced with # 10 shown in FIG. It was synthesized and ligated with T4 DNA ligase together with # 1 to # 4 to prepare a 76 bp XhoI-HinfI fragment encoding the best type signal peptide and the N-terminal side of human EGF. Plasmid pTB370 [Taniyama et al., Journal
Of Takeda Research Laboratories (J.Takeda.R
es.Lab.), 45 , 136 (1986), and JP-A-61-8888].
b The Hinf I-Pst I fragment was isolated and its 7 '
After ligating the 6 bp Xho I-Hinf I fragment with T4 DNA ligase
After treatment with XhoI and PstI, a 0.24 kb XhoI-PstI fragment was isolated. Pst I-Sma I adapter was added to this fragment to T4DN
After A ligase ligation, treatment with Xho I and Sma I,
0.24kb encoding improved signal peptide and human EGF
The Xho I-Sma I fragment was isolated.

The plasmid pGFL735Sm obtained by improving the Xho I site downstream of the human lysozyme coding region of the plasmid pGFL735 obtained in Reference Example 3 to a Sma I site was digested with Xho I and Sma I to obtain an improved signal peptide and human The DNA fragment encoding lysozyme was removed and replaced with the 0.24 kb Xho I
The plasmid pGFE101 was obtained by inserting the -SmaI fragment.

A 0.28 kb AhaIII-XhoI fragment containing the PGK terminator was isolated from the plasmid pGLDP31RcT (described above), and this fragment was inserted into the SmaI-SalI site of the plasmid pSP64 (Riboprabe, USA). −
T (PGK) was obtained. An EcoR I-Pst I fragment containing the PGK terminator was isolated from the plasmid, and the Pst I-Sma
After adding the I adapter and the Sma I-EcoR I adapter, it was inserted into the Sma I site of the above plasmid pGFE101,
The human EGF secretion plasmid pGFE213 was obtained.

Example 1. Preparation of rat C-kinase α gene Animal cell expression vector pTB755 encoding rat C-kinase α cDNA (EP-A 251,244)
In Example 5), a 3.3 kbp DNA fragment containing a PKCα gene region was prepared by EcoRI complete digestion, and this fragment was inserted into EcoRI-digested PCU18 (Takara Shuzo). Next, this recombinant DNA was used for Nco I and Pst I, Pst I and Stu
Digested with I, each with a 140 bp Nco I Pst I fragment and 2.0 kb
A Pst I Stu I fragment of p was prepared. Of these, the former is Nc
The adapter shown in FIG. 5 was added to the oI site with T4 ligase to obtain a DNA fragment of AvrII-PstI 150 bp.

The latter includes an octamer pCGAGCTCG (New Englan
d Biolabs) with T4 ligase and digested with SacI to obtain a 2.0 kpb fragment of Pst ISacI (FIG. 5).

Example 2 Construction of Expression Plasmid-1 The yeast expression plasmid pTFE112 obtained in Reference Example 2 was digested with restriction enzymes AvrII and SacI to obtain a 9.6 kbp DNA fragment. The expression plasmid pTFE755 was prepared by inserting the 150 bp fragment and the 2.0 kbp fragment obtained in Example 1 into the 9.6 kbp fragment (FIG. 6).

Example 3 Construction of Expression Plasmid-2 pTFE755 obtained in Example 2 was digested with SalI-SacI.
The 2.2 kbp fragment was inserted into the XhoI.SacI site of pTFE112 to produce the expression plasmid pTFE756 (FIG. 7).

Example 4. Obtaining and culturing yeast transformants and PKCα
Expression of protein Using the plasmid pTFE755 or the plasmid pTFE756 obtained in Examples 2 and 3, the yeast Saccharomyces cer
evisiae NA74-3A (ρ ⁻ ) (described in Reference Example 1) was transformed to transformant Saccharomyces cerevisiae NA74-3A.
(Ρ ⁻ ) / pTFE755 (IFO10442, FERM BP-1976) and NA
74-3A (ρ ⁻ ) / pTFE756 (IFO10443, FERM BP-1977) was obtained, respectively.

Then cultures of these yeast transformants 5ml [per 1, K ₂ HPO ₄ 3g, glucose 50 g, asparagine 4g, L-histidine _{100mg, KI0.1gm, MgSO 4 · 7H} 2 O500mg, CaCl 2 · 2H 2 O
_{330mg, CuSO 4 · 5H 2 O0.4mg} , FeSO 4 · 7H 2 O2.5mg, MnSO 4 · 4H 2
_{O0.4mg, (NH 4) 3 PO} 4 · 12MoO 3 · 3H 2 O0.2mg, ZnSO 4 · 7H 2 O
3.1 mg, inositol 10 mg, thiamine 0.2 mg, pyridoxine
0.2 mg, Ca-pantothenic acid 0.2 mg, niacin 0.2 mg, biotin 0.002 mg) at 30 ° C. for 3 days with shaking culture, 0.5 ml thereof was transferred to the above-mentioned medium 4.5 ml, and 30 ° C. After culturing for 1 day, 2 ml of the culture was further added to 18 ml of a fresh medium (per 1 ml of K ₂ HPO ₄ 400 mg, sucrose 80 g, asparagine 5 g, L-histidine 300 mg, KCl 2.0 g, KI 0.1 mg, MgSO ₄ .7H ₂ O 650 mg, CaCl ・ 2
H ₂ O429mg, Garasutosu 10 g, tris - maleic acid (pH 6.
_{5) 25mM, CuSO 4 · 5H} 2 O0.4mg, FeSO 4 · 7H 2 O2.5mg, MnSO 4 .4H
_{2 O0.4mg, (NH 4) 3} PO 4 · 12MoO 3 · 3H 2 O0.2mg, ZnSO 4 · 7H 2 O
3.1 mg, inositol 10 mg, thiamine 0.2 mg, pyridoxine
0.2 mg, Ca-pantothenic acid 4.0 mg, niacin 4.0 mg, and biotin 0.040 mg), and cultured with shaking at 30 ° C. for 3 days. After 72 hours, samples were taken and centrifuged (10,000 ×
g, 10 minutes) to separate the supernatant from the cells.

The above cells (200 mg, wet cell weight) were extracted with 500 μ of extraction buffer (0.1% Tween 20, 7.5 M Urea 15 mM EDTA (ethylenediaminetetraacetic acid), 2 mM PMSF (phenylmethylsulfonyl fluoride), 0.2 mM APMSF (paraaminophenylmethanesulfonyl) Fluoride hydrochloride), 5 mM EGTA (ethylene glycol-bis- (β-aminoethyl ether) N, N, N'-tetraacetic acid), 0.1 M phosphate buffer, pH
7.2), 1 g of glass beads were added, the cells were disrupted using a vortex mixer (manufactured by Scientific Industries) at 4 ° C. for 30 minutes, and then centrifuged (12,000 rpm
Minutes) and the supernatant was collected. An equal volume of SDS-gel sample buffer [VK Leammli et al., Nature (N.
227 , 680 (1970)] and heating at 100 ° C. for 10 minutes, followed by 10% SDS-polyacrylamide gel electrophoresis of the solubilized protein, followed by a known Western blotting method. [H. Towbin et al., The Prossings of the National Academy of
Sciences Proc, Natl Acad, Sci.USA) 76 , 4350
(1979)], the protein was transferred onto a nitrocellulose membrane. Known anti-PKCα monoclonal antibodies (MCClone et al.,
The expression of the PKCα protein was searched using Ashamam Co., Ltd.), and the presence of the PKCα protein was confirmed where the molecular weights were well matched.

For the band obtained by the Western blotting, PKCα protein was quantified using a densitometer, and Saccharomyces cerevisiae NA74-3A
In the case of (ρ ⁻ ) / pTFE755, the amount was about 36 μg per 1 ml of the medium. In a similar manner, Saccharomyces cerevisiae
When the protein was quantified in the case of NA74-3A (ρ ⁻ ) / pTFE756, it was about 3 μg / ml of the medium. These results indicate that the protein was produced in significant amounts by the transformed yeast.

Example 5: Measurement of PKCα activity Cells obtained by culturing according to the method of Example 4 (200 m
g, wet cell weight) 20% glycerol, 1 mM APMSF, 5 mM
EGTA, suspended in 500 μM 0.1 M Tris-HCl buffer (pH 7.5), added with 1 g of glass beads, crushed, and centrifuged (12,0
(00 rpm for 10 minutes) to collect the supernatant. Using this supernatant, the activity of protein kinase C was determined by the method of Yoshikawa et al. [U.
awa et al., Journal of Biological Chemistry (J. Biol, Chem.), 257 , 13341 (1982).
^++- dependent protein kinase activity against histone type I was confirmed. In the control in which PKCα protein was not expressed, no protein kinase activity was observed.

Effects of the Invention According to the present invention, C-kinase can be expressed in large amounts in yeast, so that C-kinase can be efficiently expressed on a large scale.
Kinase can be produced.

[Brief description of the drawings]

FIG. 1 shows a method for preparing an expression vector pTFE112. Fig. 2 shows the DNA of the improved egg white lysozyme signal peptide.
FIG. 3 shows the DNA sequence of the multilinker, and FIG. 4 shows the signal peptide of FIG.
It is a DNA sequence diagram. FIGS. 5 to 7 show the process of constructing various plasmids. FIG. 5 shows Pst encoding PACα gene.
FIG. 6 shows the construction of a 2.0 kbp fragment of I.SacI.
FIG. 7 shows the construction of pTFE756.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 9/12 C12R 1: 865) (56) References WO 88/1303 (WO, A1) (58) Fields surveyed ( Int.Cl. ⁶ , DB name) C12N 15/54 C12N 9/12 C12N 1/19 BIOSIS (DIALOG) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. Saccharomyces cerevisiae (Saccharomy)
ces cerevisiae) NA74-3A (ρ ⁻ ) / pTFE755 (FERM BP
-1976) or Saccharomyces cerevisiae
ces cerevisiae) NA74-3A (ρ ⁻ ) / pTFE756 (FERM BP
-1977), a protein kinase expression plasmid containing a DNA encoding a protein kinase C protein and capable of autonomous replication in yeast. 2. A yeast carrying the expression plasmid according to claim 1. 3. The yeast according to claim 2, wherein the yeast is a strain deficient in respiratory function. 4. The yeast according to claim 2, wherein the yeast is Saccharomyces cerevisiae. 5. Saccharomy cerevisiae (Saccharomy)
ces cerevisiae) NA74-3A (ρ ⁻ ) / pTFE755 (FERM BP
The yeast according to claim 2, which is represented by -1976). 6. Saccharomy cerevisiae (Saccharomy)
ces cerevisiae) NA74-3A (ρ ⁻ ) / pTFE756 (FERM BP
-1977). 7. A method for producing the yeast according to claim 2, wherein the yeast is cultured in a medium, and the protein kinase C protein is produced and accumulated in the cells.