WO2008020701A1

WO2008020701A1 - A new strain highly producing pdgf-bb

Info

Publication number: WO2008020701A1
Application number: PCT/KR2007/003888
Authority: WO
Inventors: Do Young Yum; Min Woo Moon; Do Young Kim; Bon Tag Koo; Chun Hee Kim; Seong Ae Kim; Min Jeong Gang; Hong Muk Yun
Original assignee: In Bionet Co., Ltd
Priority date: 2006-08-14
Filing date: 2007-08-14
Publication date: 2008-02-21

Abstract

The present invention relates to a method of increasing the expression and production efficiency of a human platelet-derived growth factor (PDGF), which is difficult to produce recombinant Iy. Specifically, the present invention aims to discover trans-secretion factors, which assist in the folding and extrascellular expression of PDGF, which is used as an agent for healing wounds such as diabetic foot ulcer, in a process of producing PDGF using a yeast expression system, and ultimately to increase the production of PDGF. The use of the present invention can reduce production cost, establish highly efficient secretion systems for a variety of high-value-added protein products, and provide biogeneric products having competitive power.

Description

[DESCRIPTION]

[Invention Title]

A NEW STRAIN HIGHLY PRODUCING PDGF-BB

[Technical Field]

The present invention relates to a method of discovering optimal secretion factors, which can increase the production of a PDGF (platelet- derived growth factor)-B homodimer difficult to produce recombinant Iy, in a process of producing PDGF using a yeast expression system, and to the use of the secretion factors for increasing the production of PDGF-B.

[Background Art]

Saccharomyces cerevisiae is a GRAS (generally recognized as safe) microorganism, which is used not only in the production of liquors or bread, but also as a raw material for various useful substances, including proteins, nucleic acids, enzymes, lipids, vitamins and minerals(Roman etc, Food Biotechnology, 1992,6:225) . Also, this microorganism is easily genetically manipulated, and various systems (e.g., expression vectors) of using it to produce foreign proteins, particularly eukaryotic cell-derived proteins like human-derived proteins, have been well constructed. Furthermore, this microorganism has an advantage in that it can perform protein secretion, glycosylation and post-translation modification, like eukaryotic cells, and thus it is used mainly in the expression and production of eukaryotic cell- derived proteins, which are difficult to produce recombinant Iy in E. coli.

The PDGF-A gene is located on chromosome 7 and has an amino acid homology of 56% with the PDGF-B gene. The PDGF consists of PDGF-A and PDGF-B chains and occurs as three isoforms: PDGF-AA, PDGF-AB and PDGF-BB(Hammacher etc, J.Biol.Chem, 1988, 263). The PDGF is known to be synthesized and released from platelets, but is currently reported to be released from various cells(Canalis etc, Academic Press Inc. U.S.A. 1996). Recently, PDGF- C and PDGF-D chains have been reported and studied(Heldin etc, Arch. Biochem. Biophys, 2002, 398:2). The PDGF is a regulatory protein having an isoelectric point of 9.8 and a molecular weight of 30 kDa, and it is known that the PDGF has a biological function of inducing the division and differentiation of smooth muscle cells and fibroblasts and is physiologically involved in tissue damage repair, inflammation, tumor formation, etcCHeldin and Westermark, Cell, regul . , 1, 1990 ;Bowen-Pope etc, Trends Genetics, 7,1991). Furthermore, it was clinically demonstrated that the PDGF has excellent effects on the healing of burn wounds and the treatment of diabetic foot ulcers, and it was found that, among various growth factors involved in wound healing, only the PDGF increased wound healing in vivo. Among different PDGF isomers, the most potent isomer is known to be the PDGF-BB homodimer, and the PDGF-BB (82-190 amino acids) has been used mainly in chronic wound healing, because it is commonly expressed on cell surfaces(J. Trauma, 1996, 41, Science, 1988, 240). As PDGF gel, becaplermin (commercially available under the trade name Regranex) is an expensive product, which depends on imports and is marketed in Korea at a price of 370,000 Won in Korean currency per 15 g (PDGF content of 0.01%). In Korea, an EGF (epidermal growth factor) spray is the sole product, but the effect of the PDGF on the treatment of foot ulcers was proved worldwide. Because of this industrial importance, there have been many efforts to increase the PDGF productivity, but the production of the PDGF is still very low. PDGF-BB was prepared by expressing and secreting it in Saccharomyces cerevisiae, was but it was mostly present in cells even after culture, and the partial

32 degradation of the protein in Arg occurred (US 4,769,328). To prevent this degradation, the protein was substituted with R28S and R32P and, as a result, it showed increased expression levels and biological activity compared to wild type, but the protein was still not well secreted. To date, various studies on secretion factors have been conducted to increase the secretion of the recombinant protein in Saccharomyces cerevisiae. The studies can be broadly divided into studies on trans-chaperones for promoting the folding of proteins newly synthesized in reticula, and studies on cis-protein fusion factors for preventing overexpressed proteins from agglomerating in reticula. Proteins of promoting the secretion and growth of proteins in yeasts include foldase and chaperone . In normal cel l cul ture , such mediator proteins are not def icient , but in recombinant strains , foreign proteins are expressed in large amounts , and thus the mediator proteins can be bott le-necked. For this reason, a method of overexpressing the protein folding-mediat ing foldase and chaperone using host technology of increasing the secret ion of recombinant proteins i s known as shown in Table 1 below , [table 1]

Protein Category Function Gene

Translocation,

Bip Chaperone Folding, Quality control

CeM p Chaperone Folding, Translocation CER I

Protein disulfide

Foldase. isomerase and PDI Disulfide formation CPR2/4/5.FPR2 Chaperone related proteins

Interconversion of

P eptidyl- prolyl

Foldase peptidyl-prolyl imide CNEI cis-trans-isomerase bonds

Chaperone,

Calne>dn Folding of glycoprotein CALRChuman) Lectin

Chaperone, Calreticulin Lectin Folding of glycoprotein

A chaperone binds to an immature protein when there is a problem in folding the protein to have a three-dimensional structure, and in addition, it is applied to induce the expression of a foreign protein in a normal intracellular process of inducing the expression of additional chaperones and foldases. When a foreign protein is not correctly folded during the production thereof, the production of the protein will be reduced, and there are many reports showing that this problem was solved by overexpressing helper proteins such as chaperones in cells. However, as shown in Table 2, a number of examples, in which the overexpression had no effects or showed adverse effects, in the case of particular target proteins, were also reported. [table 2] chaperon positive effect negative effect or non-effect

ScF v( 2 3 times).

Factor VIII, Ig, thaumatin,

Bιp(KΛR2) prochymosιn(20 times), cutinase

PDGFC 1 0 times)

FoldaseCPD Acιd-phosphatase(4 Glucoamyiase, lysozyme, I) tmes) G-CSF. IL- 1 0. TNF receptor

Hem op eroxi da se(5

Calnexπ IL-6 times)

Polyubiquiti Elafin(7 times), serum Mating factor, n albumιπ(8 times) acid-phosphatase α-amylase(2 4 times),

HAC 1 ιnvertase(tvvice) Eπdoglucanase

Accordingly, it can be seen that the effects of chaperones developed to promote the secretion of proteins as described above are different depending on the kind of proteins. This is because systems for secreting and producing proteins, developed based on traditional physiology to date, cannot be commonly applied to all proteins, and there are a number of proteins, which are impossible to recombinantly produce only with the existing systems. In order to efficiently solve this problem, there is an absolute need to develop secretion factors such as tailor-made chaperones according to the structures and characteristics of target proteins, such that the existing yeast recombinant protein secretion systems can be greatly improved. There is still no report on the experiment and analysis of secretion factors, which assist in effectively producing a PDGF-BB homodimer.

Accordingly, the present inventors have detected secretion factors such as a tailor-made chaperones, which assist in the production of the PDGF- BB homodimer, from human genes, and have demonstrated the effects thereof.

[Disclosure]

[Technical Problem]

It is an object of the present invention to develop a system of overexpressing and secreting PDGF using a PDGF secretion factor, a kind of human protein, in yeast. Another object of the present invention is to establish a yeast secretion system for the overproduction of a recombinant protein, which can express a target protein having the desired structure and characteristics and can be industrially applied because it shows reduced production cost. When a secretion system suitable for a target protein is developed, the production cost of the target protein will be greatly reduced, and it will be possible to develop a system of secreting a large amount of a high-value-added human platelet-derived growth factor as aimed in the present invention, establish systems for the highly efficient secretion of a variety of high-value-added protein products, develop systems for the high secretion of recombinant proteins for human drugs using domestic technology, and acquire intellectual property rights for each of secretion fusion factors. Also, the expression and secretion system for producing large amounts of proteins using yeast can be used to analyze not only gene base sequences, secured in the genome projects of various organisms, but also the structures and functions of protein materials, and is useful for the mass production of products, which will be medically and industrially important in future. [Technical Solution]

In order to develop a bacterial strain capable of producing PDGF-B with high efficiency, the present inventors have attempted to develop an innovative secretion system by discovering and optimizing various secretions, involved in protein secretion procedures, at the gene level through ultrahigh-speed search.

First, primers were constructed based on the prior known human PDGF base sequence, and cDNA libraries of various tissues were used to perform PCR in order to find tissues having a high PDGF expression level. As a result, a PDGF gene could be isolated from a cDNA library of kidneys. Using the isolated PDGF gene as a template, the PDGF-B(WM) gene, a PDGF-B mature gene, was amplified again and cloned into yeast expression vector YEG α- HIR525(Galactose-induced secretion vector, Choi etc, Appl , . Microbiol. Biotechnol., 1994, 42). The cloning was confirmed by extracting plasmid DNA and determining the base sequence of the extracted DNA. The recombinant protein thus prepared was named "pYGMF-PB" which was then transformed into a yeast host cell Y2805ΔgaIl strain, and the transformation was confirmed by extracting plasmid DNA and determining the base sequence of the extracted DNA. Although the vector contained a mating factor alpha secretion signal (MF leader), PDGF-BB was not easily extracellularIy secreted as expected. A yeast expression system has an advantage in that it can effectively express human genes, but it shows a great change in secretion productivity depending on the kinds or characteristics of proteins, and for this reason, it was absolutely required to introduce a tailor-made expression and secretion system in accordance with the characteristics of a target protein. Thus, secretion factors such as a trans-chaperone, which can increase the expression of human PDGF-BB, were introduced. The investigation and detection of secretion factors such as chaperones, which can increase the expression of PDGF-BB, were conducted using a human kidney cDNA library. A yeast Y2805A£?a/i/pYGMF-PB strain was transformed with a cDNA library (purchased from Clontech Lab.). The resulting transformant was cultured, and the transformation was confirmed by dot blotting and enzyme-linked immunosorbent assays. It could be seen that, when PDGF was expressed with the transformant, the expression level could be increased compared to the prior case where PDGF-BB was expressed alone. The overall flow of the present invention is shown in FIG. 1.

Meanwhile, producing the protein PDGF-B means producing the homodimer PDGF- BB, and thus "PDGF-B" and "PDGF-BB" will be interchangeably used herein.

[Advantageous Effects]

According to the present invention, an optimal secretion factor is used for the overproduction of PDGF, thus making it possible to express a large amount of PDGF-B. Accordingly, the present invention enables production cost to be reduced and is very useful for establishing systems for the highly efficient secretion of a variety of high-value-added protein proteins.

[Description of Drawings]

FIG. 1 is a schematic flow diagram showing a process of constructing a yeast transformant according to the present invention.

FIG. 2 is an electrophoresis photograph of a PDGF-B gene, amplified by PCR using, as templates, a cDNA library derived from each of human tissues, and an HELA cell library.

FIG. 3 is an electrophoresis photograph of a PDGF-B mature gene, amplified by PCR using the amplified PDGF gene as a template.

FIG. 4 is a map of a pYGMF-PB vector prepared by cloning a PDGF-B(WM⁾ gene into yeast expression vector YEG7HIR525.

FIG. 5 is a photograph of SDS-PAGE analysis, which shows that PDGF-B, produced by pYGMF-PB-transformed yeast, is not secreted in the body of yeast.

FIG. 6 shows a photograph of SDS-PAGE analysis, which shows the expression of the PDGF-B gene in pYGMF-PB-transformed yeast.

FIG. 7 is a photograph of Western blot analysis conducted to examine a change in the expression of a PDGF-BB homodimer using a mouse anti-human PDGF antibody and goat anti-mouse IgG-AP.

FIG. 8 is a photograph showing the results of qualitative analysis for the expression and secretion levels of PDGF-BB in a transformant .

FIG. 9 is a graphic diagram showing the results of tests for the PDGF- BB production of a double transformant Y2805^£a/i/pYGMF-FB/pS-26 strain according to the present invention. [Best Mode]

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are illustrative only, and the scope of the present invention is not limited thereto.

Example 1: Isolation of PDGF-B(WM) gene from human cDNA library and preparation of expression vector

(1) Isolation of PDGF-B(WM) gene

In order to examine tissues in which PDGF is largely expressed, PCR was performed using human brain, liver, kidney tissue cDNA libraries and an HELA cell library, a kind of cell line. The PCR was performed using FPDGF(5'-ATGAATCGCTGCTGGGCGCTCTTC-S'; SEQ ID NO. 1) and RPDGF(5'- CTAGGCTCCAAGGGTCTCCΓTCAG-3'; SEQ ID NO. 2) as primers for 30 cycles of 30 sec at 95 ⁰C, 45 sec at 60 °C and 30 sec at 72 ⁰C. The PCR products were electrophoresed to observe the bands (see FIG. 2). In FIG. 2, lane M is a size marker, lane 1 is a human brain-derived cDNA library, lane 2 is a human liver-derived cDNA library, lane 3 is a human kidney-derived cDNA library, and lane 4 is an HELA cell-derived cDNA library. As can be seen in FIG. 2, the PDGF-B was expressed in the human kidney-derived cDNA library.

Electrophoresis was performed using an agarose gel extraction kit (Solgent Co. Ltd) and, as a result, the PDGF-B gene of the human kidney- derived cDNA library was extracted from the agarose gel. The obtained DNA was ligated into a pGEM T-easy cloning vector at 16 ^°C for 4 hours, and then transformed into an E. coli DH5 α strain. Among the obtained E. coli colonies, 5 colonies were randomly selected, amplified by PCR using a set of primers of SEQ ID NOS: 1 and 2, and then electrophoresed to confirm whether the gene was cloned. The recombinant protein introduced with the PDGF-B gene was named "pPDGF-WF" . A mature PDGF-B gene product (see FIG. 3), amplified by PCR using the pPDGF-WF as a template with primers FPDMF(5'-AAT TCT AGA ATG AAT CGC TGC TGG GCG CTC TTC-3¹ ; SEQ ID NO: 3) and RPDMF(5'-AAT GTC GAC CTA GGC TCC AAG GGT CTC CTT CAG-3' ; SEQ ID NO: 4), was named "PDGF-B(WM)" and the base sequence thereof was analyzed (SEQ ID NO: 5). FIG. 3 shows the PDGF-B mature gene, amplified by PCR using the amplified PDGF gene as a template, and the amplified mature gene product was named "PDGF-B(WM:Wild mature)" In FIG. 3, lane 1 represents DNA derived from the E. coli DH5 α strain transformed with the human-derived PDGF-B gene.

(2) Preparation of PDGF-B(WM) gene expression vector For the cloning of PDGF-B(WM), 30 μi of yeast expression vector YEGQ- HIR525 was mixed with 6 μi of 1OX BSA, 6 μi of buffer, 3 μi of Brf I and 3 μi of Sal I, and the solution was adjusted to a final volume of 60 μJl, and then allowed to react at 37 °C for 5 hours, followed by electrophoresis. An about 5.6-kb fragment obtained by electrophoresis, and an about 980-bp fragment, containing a GALlO promoter and a mating factor-alpha secretion signal, were extracted and purified on the respective gels, thus securing DNA. Among them, 20 μi of the 980-bp fragment DNA was mixed with 6 μi of buffer, 6 μi of Xba I and 6 μi of 1OX BSA, and the solution was adjusted to a final volume, and then allowed to react at 37 ^°C for 5 hours and electrophoresed. An about 760-bp fragment was extracted and purified from the gel, thus securing DNA. 30 μi of the PCR-amplified PDGF-B(WM) DNA was mixed with 6 μi of buffer, 3 μi, of Sa/ 1 , 3 μi, of Xba I and 6 μi of 1OX BSA, and the solution was adjusted to a final volume of §Qμi,, and then allowed to react 37 °C for 5 hours and electrophoresed. Then, an about 300-bp fragment was extracted and purified from the gel, thus securing DNA.

The prepared 5.6-kb, 760-bp and 300-bp DNA fragments were subjected to three-piece ligation. Each of the DNA fragments was mixed with 3 μi of ligation buffer and 3 μi of T4 DNA ligase, and the solution was adjusted to a final volume of 30 μi, and then allowed to react at 16 °C for 16 hours.

After completion of the reaction, the ligation mixture was mixed with E. coli DH5α competent cells, followed by electroporation. Then, the mixture was mixed with LB (1% NaCl, 1% tryptone, 0.5% yeast extract) and cultured at 37 ^°C for 1 hour. Then, the culture medium was sprayed on an ampici11in-containing LB plate, thus obtaining colonies. The obtained colonies were randomly screened, and plasmid DNA was isolated and the base sequence thereof was determined to confirm whether the PDGF-B(WM) gene was cloned. The isolated plasmid DNA was named "pYGMF-PB" . A map of the major part of the expression vector pYGMF-PB is shown in FIG.4.

Example 2: Test of expression of PDGF-BB protein in veast X2&')5Λgall as host

(1) Construction of transformant yeast

To make only Y2805ΔgaJl competent cells, an Y2805ΔgaJl single colony was inoculated into 3 ml of YPD (2% pepton, 1% yeast extract), and then cultured at 30 "C overnight. The cultured seed culture medium was inoculated into 50 ml of YPD, and then cultured at 30 ⁰C until the OD₆₀O value reached about 0.4-0.6. The culture medium was centrifuged at 3000 rpm for 5 minutes. The centrifuged pellets were suspended in 25 ml of IX TE/LiAc, and then centrifuged at 3000 rpm for 5 minutes. Then, the pellets were suspended in 500 μi of IX TE/LiAc, and the suspension was dispensed in an amount of 100 μi each time and stored at 4 "C.

10 μi of the pYGMF-PB plasmid DNA, 100 μi of the competent cells, 10 μi of salmon sperms and 600 μi of PEG/LiAc were mixed with each other, and the mixture was allowed to react at 30 ^°C for 30 minutes. Then, 70 μi of DMSO was added thereto and heat-shocked at 42 ^°C for 15 minutes. The resulting material was centrifuged at 3000 rpm for 5 minutes, and the pellets were suspended in 200 μi of distilled water. The suspension was spread on a Ura-plate and cultured at 30 °C for 2-3 days, thus obtaining transformants.

(2) Analysis of expression of PDGF-BB protein

The transformed colonies were randomly screened, and then inoculated in an YPDG (2% pepton, 1% yeast extract, 1% galactose) medium and cultured for 48 hours. The culture medium was divided into a disrupted cell extract and culture supernatants for tests. In the preparation of the disrupted cell extract, the cell culture medium was measured for the OD value to adjust the amount of the cells, and then centrifuged, followed by stirring in 100 μi of Tris/PMSF. The stirred cell solution was centrifuged at 12000 rpm for 5 minutes, and the supernatant was removed. To the pellets, 30 μi of Tris/PMSF and the same amount of glass beads were added, followed by stirring. The stirred solution was mixed with 200 μi of Tris/PMSF and centrifuged at 12000 rpm for 5 minutes. The supernatant was modified with 5X sample buffer and subjected to SDS-PAGE (see FIG. 5). In FIG. 5, lanes 1-4 represents the yeast Y2805Δgall culture supernatant, the yeast Y2805A§a/i/YEGα-HIR525 culture supernatant, the yeast Y2805-4,g3/i/pYGMF-PB culture supernatant and the yeast Y2805Λ?a/i/pYGMF-PB disrupted cell extract.

Whether the bands shown in FIG. 5 were caused by the PDGF-B(WM) protein was examined through an immune response method. For this purpose, a change in the expression of the PDGF-BB homodimer was analyzed by Western blotting using a mouse anti-human PDGF antibody and goat anti-mouse IgG-AP.

The culture supernatants were quantified for the protein using the Bradford method, and then added to the same amount of 100% cold acetone and left to stand on ice for 30 minutes. After this, the solutions were centrifuged at 4 "C and 13000 rpm for 15 minutes, and the pellets were washed with 70% EtOH, and then suspended in IX PBS and concentrated. Like the disrupted cell extract, the concentrates were modified with 5X sample buffer and subjected to SDS-PAGE. After development on the SDS-PAGE gel, the concentrates were stained with Coomassie blue to observe the bands, and then transferred to an NC (nitrocellulose) membrane and blocked with blocking solution (5% skim milk in IX TBS) at room temperature for 2 hours. After removal of the blocking solution, a primary antibody (Mouse Anti-PDGF, monoclonal), dissolved in blocking solution, was added thereto at a ratio of 1:2000, and the membrane was incubated with shaking incubation at 4 ^°C overnight. The shaking-incubated membrane was washed three times with TTBS (0.05% Tween 20 in IX TBS) for 10 minutes each time. Then, a secondary antibody (anti-mouse IgG) was added thereto at a ratio of 1:4000, and the membrane was incubated with stirring at room temperature for 2 hours. The membrane was washed with TTBS and developed in a detection buffer, containing NBT and BCIP, in a dark place. Western blotting was conducted using an anti- human PDGF antibody to examine the change in the expression level of PDGF-BB (see FIG.6). In FIG. 6, each of lanes is the same as in FIG. 5.

Example 3: Investigation of new secretion factors which increase expression and secretion of PDGF-BB

(1) Construction of double transformants for investigation of secretion factors

To screen secretion factors capable of increasing the expression of PDGF, a human kidney cDNA library (Cat. HL4043AH) , which is mesodermal- derived tissue from which human PDGF has been separated, was purchased from Clontech for use in experiments. The cDNA library was cloned into an EcoR \Aho I site in a pACT2 vector and had an insert size of 0.5-4.0 kb. Hereinafter, for the convenience of description, an empty vector having no human kidney cDNA library introduced therein will be expressed as pACT2-, and a vector introduced with the human kidney cDNA library will be expressed as pACT2.

The cDNA library was transformed into E. coli DH5 α and then serially diluted and spread on an ampicil 1 in-containing LB plate. The appropriate dilution factor of the cDNA library was determined, and on the basis of the determined dilution factor, the cDNA was spread on the plate to obtain a large amount of a transformant from which plasmid DNA was then isolated.

Y2805ΔgaJl (Ura, Leu auxotroph) as a host cell, cloned into the pACT2 vector containing an Ieu2 gene, was transformed with PDGF-B(WM) cloned into a ura gene-containing YEGα-HIR525 vector, that is, pYGMF-PB. The competent cell of the yeast Y2805z\_£a/i/pYGMF-PB strain was prepared in the same manner as mentioned above.

10 μJt of the human kidney cDNA library, 100 μl of the Y2805Z\ _£a/i/pYGMF-PB strain competent cell, 10 μl of salmon sperm DNA and 600 μJt of PEG/LiAc were mixed with each other and left to stand at 30 ⁰C for 30 minutes. Then, 70 μl of DMSO was added thereto and the mixture was heat- shocked at 42 °C for 15 minutes. The heat-shocked material was centrifuged at 3000 rpm for 5 minutes, and then suspended in 200 μi of distilled water. The suspension was spread on a YG (YNB & Glucose, UL-) plate and cultured at 30 °C for 2-3 days, thus obtaining double transformant colonies.

(2) Qualitative analysis of expression and secretion of PDGF-BB in double transformants

In order to culture large amounts of the colonies in the same condition, a 96-deep-well plate was used, because yeast would not well grow on a general 96-well plate. 600 μl, of YPDG (1% yeast extract, 2% peptone, 1% dextrose, 1% galactose) medium was placed into each well of a sterilized 96- well plate containing glass beads, and each of the transformants was inoculated into each well. At this time, each of the transformants was also picked on a YG(YNB & Glucose, UL-) plate to obtain master cells. The inoculated transformants were placed in a 96-deep-well plate incubator (Bioneer Co. Ltd) and cultured at 30 ⁰C at 400 rpm for 48 hours with the supply of oxygen.

The cultured cells were centrifuged to remove pellets, and 50 μi of the supernatant was loaded into a dot blot kit containing an NC (nitrocellulose) membrane, and was washed with two times with 50 μi of IX TBS. The membrane was placed in a sealing bag and blocked with blocking solution (5% skim milk in IX TBS) at room temperature for 2 hours. After the blocking solution was removed, a primary antibody (mouse anti-PDGF, monoclonal), dissolved in blocking solution, was added thereto at a ratio of 1:2000, and the membrane was incubated with shaking at 4 ^"C overnight. The shaking-incubated membrane was washed three times with TTBS (0.05% Tween 20 in IX TBS) for 10 minutes each time, and then a secondary antibody (Anti- mouse IgG) was added thereto at a ratio of 1:4000. Then, the membrane was incubated with shaking at room temperature for 2 hours. The incubated membrane was washed with TTBS in the same manner as described above, and then developed in a detection buffer containing NBT and BCIP, in a dark place, to qualitatively analyze the expression and secretion of the protein (see FIG. 7).

FIG. 7 shows the results of the qualitative observation of a change in the expression of PDGF-BB in the yeast Y2805Aga/i/pYGMF-PB strain transformed with the human kidney-derived cDNA library, in which the qualitative observation was conducted using the dot blot technique to primarily detect secretion factors capable of increasing the expression of a mature PDGF-B(WM) gene in the yeast Y2805^^a7i/pYGMF-PB strain. In FIG. 7, Nos. 1, 2 and 3 indicate control groups. Specifically, No. 1 indicates the culture supernatant of yeast Y2805^^a/i/pYGMF-PB/pACT2-double transformant (one doubly transformed with empty pACT2 have no human kidney-derived cDNA inserted therein), No. 2 indicates the culture supernatant of yeast Y2805Z., £a/i/pYGMF-PB, and No. 3 indicates the culture supernatant of yeast Y2805Z\ gall. Unnumbered blots in FIG. 7 indicate the culture supernatant of the yeast Y2805Aga/i/pYGMF-PB strain doubly transformed with pACT2 having human kidney-derived cDNA introduced therein, that is, Y2805^^a/i/pYGMF-PB/pACT2. Samples having a signal intensity higher than those of the three controls were primarily selected.

Example 4: Quantitative analysis of expression and secretion of PDGF- BB in double transformants and selection of transformants

82 colonies, which showed high PDGF-BB signals in the primary investigation conducted using the dot blot technique, were primarily analyzed by ELISA (data not shown). The colonies were examined for the master cells and secondarily analyzed by ELISA. The ELISA process was performed using a kit including a human PDGF-BB DuoSet ELISA development system (Cat. DY220, R&D System).

Each of the double transformants was inoculated into 5 ml of YPDG (1% yeast extract, 2% peptone, 1% dextrose and 1% galactose), and then cultured at 30 °C for 48 hours at 180 rpm.

In order to perform ELISA, 100 id of a dilution of a capture reagent (recombinant human PDGF Rβ was allowed to react with each of the transformants on each well of a coating plate at room temperature overnight. After the solution in each well was removed, the well was washed three times with PBST (0.05% Tween20 in PBS) for 10 minutes each time, and then allowed to react with blocking solution (1% BSA in PBS) at room temperature for one hour. After this, the well was washed with PBST, and the PDGF standard in the kit and the same amount of the culture supernatant diluted to a given concentration were added thereto and allowed to react at room temperature for 2 hours. After the well was washed with PBST, a detection antibody (goat anti-human PDGF-BB) was added thereto and left to stand at room temperature for 2 hours. Then, the well was washed with PBST, and streptavidin-HRP was added thereto, and then allowed to react at room temperature in a dark place for 20 minutes. Then, the well was washed with PBST, after which a substrate solution (TMB, SIGMA) was added thereto and allowed to react at room temperature in a dark place for 20 minutes. Then, a stop solution (2N H2SO4) was added and the absorbance values at 450 nm and 540 nm were measured. To reduce measurement error, a corrected value obtained by subtracting the absorbance value at 540 nm from the absorbance value at 450 nm was used. In this manner, secondary analysis was performed three times by ELISA with the PDGF-BB standard, the measurement results were averaged, and 17 transformants showing an increase in the expression of PDGF-BB were numbered with "S". The concentrations of PDGF-BB in the culture supernatants of the 17 double transformants are graphically shown in FIG. 8. In FIG. 8, No. 1 indicates the yeast Y28Q5ΔgaIl culture supernatant, No. 2 indicates the yeast Y2805Z\ sa/i/YEGα-HIR525 culture supernatant, and No. 3 indicates the yeast Y2805Zi £2/i/pYGMF-PB/pACT2- culture supernatant.

Example 5: Sequencing of PDGF-BB secretion factors

(1) Amplification of DNA of selected strains

On the basis of the results of the secondary ELISA, three strains (pS^~ 12, pS-17 and pS-26) showing the highest PDGF-BB expressions (pS-12, 17, 26) were selected. To isolate DNA from the strains, each of the strains was inoculated into 3 ml of YPD (1% yeast extract, 2% peptone and 2% dextrose), and then cultured overnight. The cell culture medium was centrifuged at 13000 rpm for 10 minutes, and the supernatant was removed. Then, the pellets were suspended in 50 μi of STES(O.5M NaCl, 0.2M Tris-HCl (pH7.6), 0.01M EDTA, 1% SDS) buffer, followed by centrifugation. Following this, the same amount of STES buffer was added to the suspension, glass beads were added thereto, and then the solution was stirred for 5 minutes. The stirred solution was allowed to react with IX TE and RNase A (20 mg/ml) at 30 °C for 1 hour, and then a mixture of phenol, chroloform and isoamyl alcohol (25:24:1) was added thereto, followed by stirring for 5 minutes. The stirred solution was centrifuged at 13000 rpm for 10 minutes, and the supernatant was transferred into a fresh tube and precipitated with 3M sodium acetate and 100% EtOH. The precipitate was washed with 70% EtOH, dried, dissolved in distilled water, and electrophosed to confirm the DNA of the strains.

1 μi of the DNA, 2 μi of primer FSCR:5'- CTATTCGATGATGAAGATACCCCACCAAAC-3'(SEQ ID NO: 6) (10 pmol), 2 μi of primer RSCR: 5'-TGM(TKK)(X^TTTTTCAGTATCTACGA-S' (SEQ ID NO: 7) (10 pmol) and Aμi of 5X PCR premix were mixed with each other. The mixture was adjusted to a final volume of 20 μi, and then subjected to PCR for 30 cycles of 1 min at 95 "C, 1 min at 55 °C and 3 min at 68 ⁰C. The PCR product was electrophoresed to determine the insert size. 3 μi of the PCR product was used for transformation.

The E. coli DH5 α competent cells were mixed with the above-obtained plasmid DNA and electroporated. Then, the cells were cultured in LB, and then spread on an LB plate containing ampicillin, thus securing transformants.

(2) Sequencing of PDGF-B section factors

The above-secured transformants were randomly screened and were inoculated into LB containing ampicillin. Then, the plasmid DNA was isolated from the transformants, and the base sequence thereof was analyzed.

From three consistently positive clones among candidates obtained by back-transforming the YY2805A£3/i/pYGMF-PB strain, plasmid DNA was isolated and purified. Then, the base sequences thereof were analyzed and, as a result, it could be seen that genes having three different sequences were inserted into the plasmid DNA and acted as protein secretion factors (Table 3).

[table 3]

Accession Gene size BLAST analysis a^Smi number (bp) (SEQ ID No. )

_ _{H O} BC0070651 . 1 _{H n}__ Glutathione S-transf erase T 1 -1

Po-1 -i 10Yb

Gl: 13737910 (SEQ ID No. 8) n_{ς 1}7 _Rrnc_{ififi i} n _? n w unnamed protein product pS-17 BC036610.2 1 152 (_SEQ ID No. 9)

DQ246833 cytochrome c oxidase subunit I

^{P ~} Gl 78499271 (SEQ ID No. 10)

Glutathione S-transferase (GST) contained in pS-12 is known to be a protein, which is used for fusion wi th a fusion protein to be puri f ied, in order to faci l itate the puri f icat ion of the protein. However , there is no report showing that pS-12, pS-17 and pS-26 assist in the secretion and production of proteins such as PDGF.

Example 6: Analysis of production of PDGF-BB in 5-liter fermenter Among the screened strains, the Y2805Λ§tf/i/pYGMF-PB/pS-26 strain was cultured at high concentration in a 5-liter fermenter in order to analyze the possibility of industrial application of PDGF-BB.

The strain was cultured in a seed culture medium, containing 2% glucose, 1% yeast extract and 2% bactopeptone, for 20-24 hours. Then, the strain was cultured in a medium, containing 2% glucose, 4% yeast extract and 1.5% bactopeptone, for 72 hours. In the culture process, a fed-batch culture process was carried out at a shaking speed of 500-900 rpm with an aeration of 1.0-2.0 wm (v/v). From the point of time when glucose in the medium was almost exhausted after 13 hours, the medium was replaced with a fresh medium (60% glucose and 5% yeast extract), and galactose induction (1.5% galactose based on the total volume) was carried out after 40 hours at which the measured OD_60O was more than 100. The results of three experiments showed that

PDGF-BB could be produced at a concentration of 115-130 ng/ml after 54-72 hours (see FIG. 9(a)-(c)). [Industrial Applicability]

As described above, the present invention enables PDGF-B to be expressed in a large amount. Thus, the use of the present invention can reduce production cost and is very useful for establishing systems for the highly efficient secretion of a variety of high-value-added protein products.

Claims

[CLAIMS] [Claim 1]

Recombinant yeast which produced and secreted PDGF-BB to the extracellular with high efficiency after carryingCor harboring) genes encoding heterologus platelet-derived growth factor B (PDGF-B) and secretion- associated protein by co-transformation.

[Claim 2]

The yeast of Claim I₁ wherein both PDGF-B gene and secretion- associated gene are cloned on different expression vectors.

[Claim 3]

The yeast of Claim 1 or 2, wherein the secretion-associated gene is derived from a human kidney cDNA library.

[Claim 4]

The yeast of Claim 1 or 2, wherein the secretion-associated gene is a homologue of cytochrome oxidase subunit I (CCOS I).

[Claim 5]

The yeast of Claim 1 or 2, wherein the gene encoding the secretion- associated protein is a homologue of GST (glutathione S^~transferase).

[Claim 6]

The yeast of Claim 1 or 2, wherein SEQ ID NO-- 9 represent a gene encoding the secretion- associated protein.

[Claim 7]

The yeast of Claim 1 or 2, wherein the recombinant plasmid pYGMF-PB is the vector with PDGF-B gene as shown in FIG. 4 and Plasmid pACT2 contains a secretion- associated gene.

[Claim 8]

The yeast of Claim 7, which is recombinant strain Y2S05Δga11/pYGW- PB/pACT2.

[Claim 9]

A protein which is encoded by SEQ ID NO: 9.

[Claim 10] DNA sequence(or gene)encoding the protein of Claim 9.

[Claim 11]

A method for constructing yeast of producing and secreting PDGF-BB with high efficiency, the method comprising the steps of:

(A) isolating a platelet-derived growth factor B (PDGF-B) and cloning the PDGF-B gene into an expression vector;

(B) transforming yeast with the expression vector pYGMF-PB, culturing transformants, and isolating the primary recombinant yeast which expressed PDGF-BB; and

(C) transforming the primary recombinant yeast with a human kidney cDNA library and isolating a secondary recombinant yeast which secreted PDGF- BB to the extracellular medium.

[Claim 12]

The method of Claim 11, wherein the human kidney cDNA library in the step (c) contains a GST gene.