KR100726143B1 - - A New Strain Highly Producing PDGF-BB and a Method for Constructing the Strain - Google Patents

Abstract

A yeast is provided to mass-express platelet-derived growth factor(PDGF) by using an optimum secretion factor for overproduction of the PDGF, thereby capable of reducing the manufacturing cost and being useful for establishing high efficiency secretion system of various high valued protein products. The yeast producing and secreting PDGF-B with high efficiency is characterized in that it is co-transformed by a PDGF-B gene and a secretion factor gene derived from human kidney. In the yeast, the PDGF-B gene is existent in a vector pYGMF-PB and the secretion factor gene is existent in a vector pACT2. The yeast is Y2805Deltagall//pYGMF-PB/pACT2. The method for preparing the yeast comprises the steps of: (a) after isolating and cloning the PDGF-B, introducing it into an expression vector; (b) after transforming yeast with the expression vector, culturing it and then isolating a first transformed yeast in which the PDGF-B is expressed; and (c) after transforming the first transformed yeast with a human cDNA library, isolating a second transformed yeast in which the PDGF-BB is extracellularly secreted.

Description

A strain for producing and secreting a recombinant human platelet-derived growth factor (PDV-F) with high efficiency and a method for producing the strain {A New Strain Highly Producing PDGF-BB, and a Method for Constructing the Strain}

1 is a schematic flowchart showing a process for producing a yeast transformant according to the present invention.

Figure 2 is an electrophoresis picture of amplified PDGF-B gene by the PCR method using the cDNA library and HELA cell library derived from each human tissue.

Figure 3 is an electrophoresis picture of amplified PDGF-B mature gene again using the PCR technique using amplified PDGF gene as a substrate.

Figure 4 is a pYGMF-PB vector map prepared by cloning the PDGF-B (WM) gene in the yeast expression vector YEGα-HIR525.

Figure 5 is a photograph of the SDS-PAGE results showing that PDGF-B produced by pYGMF-PB transgenic yeast is not secreted in the body of yeast.

Figure 6 is a photograph of the SDS-PAGE results showing the PDGF-B gene expression of pYGMF-PB transgenic yeast.

Figure 7 is a photograph of Western blotting results using a mouse anti-human PDGF antibody, goat anti-mouse IgG-AP to confirm the difference in expression of PDGF-BB monomer.

8 is a qualitative analysis result photograph of PDGF-BB expression and separation amount of a transformant.

Figure 9 is a graph showing the PDGF-BB production test results of the double transformant Y2805 Δgal1 / pYGMF-PB / pS-26 strain according to the present invention.

The present invention uses PDGF (platelet-derived growth factor) -B monodimer, which is a human-derived protein, which is difficult to produce in recombinant production, to find an optimal secretion factor that can increase its productivity during production using a yeast expression system. It is about increasing productivity using methods and secretion factors.

Yeast Saccharomyces cerevisiae is a GRAS (Generally Recognized As Safe) grade that is harmless to human body used as raw materials for proteins, nucleic acids, enzymes, lipids, vitamins, minerals, etc. It is a microorganism (Roman et al., Food Biotechnology, 1992, 6: 225) and has a well-established system for the production of higher-cell-derived proteins, such as genes and related foreign proteins such as expression vectors, especially human-derived proteins. In addition, it is a microorganism mainly used for the expression and production of higher cell-derived proteins, which are difficult to produce recombinant proteins through Escherichia coli, because they have the advantages of similar protein secretion function, sugar chain addition function, and post-translational modification function.

The PDGF-A gene is located confined to chromosome 7, and the identity of the PDGF-B gene and amino acid sequence is 56%. PDGF exists in three dimeric forms, PDGF-AA, PDGF-AB and PDGF-BB by PDGF-A and PDGF-B chains (Hammacher et al., J. Biol. Chem, 1988, 263). The source of release is known as platelets, but is currently released in various cells (Canalis et al., Academic Press Inc. USA 1996). Recently, the PDGF-C chain and the D chain have been reported and researches are being actively conducted (Heldin et al., Arch. Biochem. Biophys, 2002, 398: 2). PDGF has an isoelectric point of 9.8 and is a regulatory protein with a molecular weight of 30 kDa. Biological function is known to induce the division and differentiation of striated myocytes and fibroblasts, and to be physiologically involved in the repair of tissue damage, inflammation and tumor formation. Heldin and Westermark,, Cell. Regul., 1, 1990; Bowen-Pope et al., Trends Genetics, 7,1991). Clinically, burns are known to be effective in treating wound healing and diabetic foot ulcers. Among the growth factors involved in wound healing, only PDGF enhances wound healing in vivo . The strongest isomer of the different PDGF isomers is known as the PDGF-BB monodimer, and PDGF-BB (82-190 amino acids) is commonly expressed on the cell surface and has been used extensively in the treatment of chronic wounds (J. Trauma, 1996, 41, Science, 1988, 240). Currently, PDGF gel is imported and sold in full quantity of becaplermin (trade name Regranex), and it is an expensive product sold for 370,000 won / 15g (0.01% PDGF content). Daewoong Pharmaceutical's EGF (Epidermal Growth Factor) spray is the only one in Korea, but PDGF has been proven worldwide in treating foot ulcers. Due to this industrial importance, much attention is paid to the productivity improvement of PDGF, but the production amount is very small. PDGF-BB was produced as an expression secretion in Saccharomyces cerevisiae, but was mostly intracellular after culture and partial proteolysis occurred in Arg ³² (US 4,769,328,1999). Substitution with R28S and R32P to prevent this degradation resulted in increased expression and slightly increased biological activity compared to wild-type but still not secreted well. To date, various secretion factors have been studied in Saccharomyces cerevisiae to enhance the secretory capacity of recombinant proteins. Trans-type chaperone studies that help fold newly synthesized proteins in the endoplasmic reticulum and overexpressed proteins aggregate in the endoplasmic reticulum. It can be divided into cis-type protein fusion factor studies to prevent them. Proteins that help secrete and mature secretory proteins in yeast include Foldase and chaperone. In normal cell culture, these mediators do not lack these media, but recombinant strains express large amounts of foreign proteins. Because of the potential for bottle-neck, overexpression methods of Fall Days and chaperones that mediate protein folding in host engineering to enhance secretion of recombinant proteins are shown in Table 1.

Chaperone is used to express foreign proteins as a normal intracellular process that not only binds to immature proteins but also induces the expression of additional chaperones and Fall Days if there is a problem of folding the protein properly to have a tertiary structure. When protein is not properly folded during foreign protein production, many cases have been reported to solve this problem by overexpressing helper proteins in cells such as chaperone. However, a number of examples of ineffective or rather adverse effects depending on the type of target protein were reported as shown in Table 2.

Therefore, as described above, it can be seen that the effect of chaperone developed for protein secretion shows different results depending on the type of protein. This problem is due to the fact that existing protein secretion production systems developed on the basis of traditional physiology are not universally applicable to all proteins, and there are many proteins that cannot be recombinantly produced by the existing system alone. In order to significantly improve the uniform yeast recombinant protein secretion system, it is absolutely necessary to develop secretory factors such as tailer-made chaperones according to the structure and properties of the target protein. To date, no experimental confirmation or analysis of secretion factors has been reported to help produce PDGF-BB monodimer.

Accordingly, the present inventors have detected a custom secreted secretion factor such as chaperon from the human genome to help produce PDGF-BB monodimer and prove its effect.

An object of the present invention is to develop a hyperexpression secretion system using PDGF secretion factor, a kind of human protein using yeast. In addition, by establishing a yeast secretion system for the excessive production of recombinant proteins to express the desired protein in accordance with the structure and properties, and to secure the technology that can be applied to industrialization through cost reduction. In case of developing a secretion system suitable for the target protein, the production cost is greatly reduced.In addition to the development of a mass secretion system of high value-added human platelet growth factor, various high value-added protein products can be established in Korea. Its own technology enables the development of high secretion systems of human pharmaceutical recombinant proteins and the acquisition of intellectual property rights for each secretion fusion factor. In addition, it is useful for mass production of medicinal and industrially important products in the future as well as analysis of the structure and function of genome sequences and protein materials obtained from genome projects of various living organisms using the mass production expression and secretion system using yeast. Could be used.

The present invention was intended to develop an innovative secretion system by discovering and optimizing various secretion factors involved in the protein secretion process through ultrafast search in the gene unit for the development of strains capable of producing PDGF-B with high efficiency.

First, primers were prepared based on the known PDGF sequences of humans, and PCR was performed using cDNA libraries of various tissues to find tissues in which PDGF was expressed. As a result, PDGF gene was obtained from kidney cDNA library. Using the isolated PDGF gene as a template, PDGF-B (WM) gene, a PDGF-B mature gene, was amplified again and the yeast expression vector YEGα-HIR525 (Galactose-induced secretion vector, Choi et al., Appl, Microbiol. Biotechnol., 1994, 42) was cloned (cloning) and the presence or absence of the cloning was confirmed by sequencing the plasmid DNA extracted. The recombinant thus prepared was named pYGMF-PB and transformed into Y2805 Δ gal1 strain, a yeast host cell, and the presence or absence of transformation was also confirmed by plasmid DNA extraction and sequencing. Although it contained a cross-linker alpha secretion signal (MFα leader) in the vector, PDGF-BB, as expected, did not have extra-cellular secretion. The yeast expression system has the advantage of effectively expressing human genes. However, since it shows tremendous difference in secretion productivity according to the type and characteristics of protein, introduction of customized expression and secretion system according to the characteristics of target protein is absolutely necessary. So we introduced secretory factors such as trans-chaperone that can increase human PDGF-BB expression. Human kidney cDNA library was used for the detection and detection of secretion factors such as chaperone that can increase the expression of PDGF-BB. After transforming the yeast Y2805 Δgal1 / pYGMF-PB strain with the cDNA library purchased from Clontech Co., Ltd., the transformant was cultured and then transformed through dot blot and enzyme-linked immunosorbent assay. After confirming the reproducibility of the experiment, it was possible to increase the amount of expression than expressing PDGF-BB alone when expressed with PDGF. The overall flow of the invention is shown in FIG.

On the other hand, since the production of the protein PDGF-B means the same as producing the end-dimer PDGF-BB, hereinafter, the expression of PDGF-B and PDGF-BB for convenience.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The embodiments are only listed by way of example to illustrate the present invention, thereby not changing or reducing the scope or content of the technical spirit of the present invention.

Example 1 Isolation of PDGF-B (WM) Gene from Human CDNA Library and Preparation of Expression Vector

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

PCR was performed using the human brain, liver, and kidney tissue cDNA libraries and HELA cell library, a kind of cell line, to identify tissues with high PDGF expression. Primers used for PCR were FPDGF (5'-ATGAATCGCTGCTGGGCGCTCTTC-3 '; SEQ ID NO: 1) and RPDGF (5'-CTAGGCTCCAAGGGTCTCCTTCAG-3'; SEQ ID NO: 2) at 95 ° C for 30 seconds, at 60 ° C for 45 seconds, 72 30 cycles were repeated for 30 seconds at ℃. PCR products were electrophoresed in a conventional manner to confirm the band (Fig. 2). In the figure, lane M is the size marker (hereinafter identical), 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 a HELA cell derived cDNA library. As can be seen in the figure, it was confirmed that PDGF-B gene was expressed in human kidney-derived cDNA library.

As a result of electrophoresis using agarose gel extraction kit (Solgent Co. Ltd), PDGF-B gene of human kidney-derived cDNA library was extracted from agarose gel. The obtained DNA was ligation into the pGEM T-easy cloning vector at 16 ° C. for 4 hours, and five colonies of E. coli colonies obtained by transforming E. coli DH5α strain were randomly selected to obtain a primer set of SEQ ID NOs: 1 and 2. After amplification by using a PCR technique and electrophoresis was confirmed whether cloning. The recombinant into which the PDGF-B gene was introduced was named pPDGF-WF. pPDGF-WF as a template Primer FPDMF (5'-AAT TCT AGA ATG AAT CGC TGC TGG GCG CTC TTC-3 '; SEQ ID NO: 3) and RPDMF (5'-AAT GTC GAC CTA GGC TCC AAG GGT CTC CTT CAG-3 The mature PDGF-B gene product (see FIG. 3), amplified by PCR using SEQ ID NO: 4), was named PDGF-B (WM) and analyzed for nucleotide sequence (SEQ ID NO: 5). Figure 3 is amplified PDGF gene as a substrate as a result of amplifying only the PDGF-B mature gene again using PCR technology, the amplified gene product was named PDGF-B (WM: Wild mature). Lane 1 in the Figure means DNA derived from E. coli DH5α strain transformed with human-derived PDGF-B gene.

(2) Preparation of PDGF-B (WM) Gene Expression Vector

After PDGF-B (WM) for the first cloning 30㎕ yeast expression vector YEGα-HIR525 vector, into a 10X BSA 6㎕, buffer 6㎕, B amH Ⅰ3㎕, S al Ⅰ3㎕ align the final volume to 60㎕ The reaction was carried out at 37 ° C. for 5 hours and electrophoresed. About 5.6 kb fragment obtained by electrophoresis and about 980 bp fragment containing GAL10 promoter and cross-factor alpha secretion signal were extracted and purified from gel, respectively, to obtain DNA. Among them, 20 μl of 980bp fragment DNA, 6 μl of buffer, 6 μl of X baI , and 6 μl of 10 × BSA were added to 60 μl of final volume, followed by reaction at 37 ° C. for 5 hours and electrophoresis. Here, about 760bp fragment was extracted and purified from a gel to secure DNA. PCR amplification 30㎕ a PDGF-B (WM) DNA, buffer 6㎕, S al Ⅰ 3㎕, 3 μl of X ba I and 6 μl of 10X BSA were added and the final volume was adjusted to 60 μl, followed by reaction at 37 ° C. for 5 hours and electrophoresis. After that, the fragment at about 300bp was extracted from the gel and purified to obtain DNA.

Thus prepared 5.6kb, 760bp, 300bp DNA fragments were three-pieces ligation. 3 l of each DNA, ligation buffer, and 3 l of T4 DNA ligase were added and the final volume was adjusted to 30 l, followed by reaction at 16 ° C. for 16 hours.

The reaction mixture was mixed with E. coli DH5α competent cell, electroporation, mixed with LB (1% NaCl, 1% tryptone, 0.5% yeast extract), incubated for 1 hour at 37 ℃, and then spread on Ampicillin-added LB plate. Colonies were obtained. The colonies thus obtained were randomly selected and sequenced after plasmid DNA isolation to confirm PDGF-B (WM) gene cloning and named as pYGMF-PB. The main part map of the expression vector pYGMF-PB is shown in FIG.

Example 2: Yeast Y2805 △ gal1  Expression Experiment of PDGF-BB Protein Using As Host

(1) Production of transgenic yeast

Y2805 Δgal1 single colony was inoculated in 3 ml of YPD (2% pepton, 1% yeast extract) incubated overnight at 30 ° C. to make only Y2805 Δgal1 competent cells. After incubating overnight culture of 1% in YPD 50ml incubated at 30 ℃ so that the OD ₆₀₀ value 0.4 ~ 0.6 and centrifuged at 3000 rpm, 5 minutes. Then, 25 ml of 1X TE / LiAc was added and suspended, followed by centrifugation at 3000 rpm for 5 minutes, and the pellet was again suspended in 500 µl of 1X TE / LiAc, and 100 µl was dispensed and stored at 4 ° C.

10 μl of pYGMF-PB plasmid DNA, 100 μl of Competent cell, 10 μl of Salmon sperm DNA, and 600 μl of PEG / LiAc were reacted for 30 minutes at 30 ° C., 70 μl of DMSO was added and heat-shocked at 42 ° C. for 15 minutes. After centrifugation at 3000 rpm for 5 minutes, the cells were suspended in 200 μl of distilled water, plated on a Ura-plate, and incubated at 30 ° C. for 2-3 days to obtain a transformant.

(2) Expression Analysis of PDGF-BB Protein

Transformed colonies were randomly selected and inoculated in YPDG (2% pepton, 1% yeast extract, 1% galactose) medium and incubated for 48 hours. Cell disruption extract was prepared by measuring the OD value of the cultured cells to adjust the amount of cells and centrifuged, Tris / PMSF 100μ was added and stirred. After centrifugation at 12000 rpm for 5 minutes, the supernatant was removed, and 30 μl of Tris / PMSF was added thereto, followed by stirring with an equal amount of glass beads. 200 μl of Tris / PMSF was added thereto and centrifuged at 12000 rpm for 5 minutes, followed by denaturation by mixing the supernatant with 5X sample buffer (FIG. 5). Lanes 1 to 4 in the yeast Y2805 Δgal1 culture supernatant, yeast Y2805 Δgal1 / YEGα-HIR525 culture supernatant, yeast Y2805 Δgal1 / pYGMF-PB culture supernatant and yeast Y2805 Δgal1 / pYGMF-PB cell disruption extract, respectively Indicates.

It was confirmed once again by the immunoassay whether the band identified in Figure 5 is due to PDGF-B (WM) protein. To this end, the expression difference of PDGF-BB monomer was analyzed using Western blotting technique using mouse anti-human PDGF antibody, goat anti-mouse IgG-AP.

The culture supernatant was quantified by the Bradford method and added to the same amount of 100% Cold Acetone and left on ice for 30 minutes. After centrifugation at 4 ℃, 13000 rpm for 15 minutes, the pellet was washed with 70% EtOH, suspended in 1X PBS and concentrated. SDS-PAGE was performed by denaturing with 5X sample buffer as in the cell disruption extract. After developing on the SDS-PAGE gel, the band was confirmed by staining with coomassie blue, and a blocking solution (5% skim milk in 1X TBS) was added to the transfer membrane to the NC (Nitrocellulose) membrane, and then blocked at room temperature for 2 hours. After removing the blocking solution, the primary antibody (Mouse Anti-PDGF, monoclonal) dissolved in the blocking solution was added at a ratio of 1: 2000 and shaken incubation overnight at 4 ° C. After overnight shaking incubation membrane was washed three times for 10 minutes with TTBS (0.05% Tween 20 in 1X TBS), and then the secondary antibody (Anti-mouse IgG) was added at a ratio of 1: 4000 and shaked incubated for 2 hours at room temperature. After washing the membrane with TTBS, NBT and BCIP were added to the detection buffer and developed in the dark. Western blotting using anti-human PDGF antibody confirmed the difference in the amount of PDGF-BB expression (Fig. 6). Each lane is the same as in FIG. 5.

Example 3 Exploration of new secretion factors that increase PDGF-BB expression and secretion

(1) Construction of double transformant for secretion factor search

In order to select a secretion factor that can increase the expression of PDGF, human kidney was isolated from human PDGF and Mesodermal-derived human kidney cDNA library (Cat. HL4043AH) was purchased from Clontech. The cDNA library is cloned into the E coR I / X ho I site in the pACT2 vector and the insert size is 0.5-4.0 kb. For convenience of explanation, vacant vectors into which human kidney cDNA library is not introduced are referred to as pACT2- and vectors into which human kidney cDNA library is introduced are represented by pACT2.

The cDNA library was transformed into E. coli DH5α, serial dilution was carried out on an LB plate containing ampicillin, and then the appropriate dilution factor was determined. Based on this, the plasmid DNA was recovered from a large number of transformants.

which is cloned into the YEGα-HIR525 vector to the ura gene present PDGF-B (WM) that is pYGMF-PB and cloned in pACT2 vector of the leu2 gene present human kidney cDNA library Y2805 △ capable of screening both types at once, gal1 that Transformation was performed using (Ura, Leu auxotroph) as a host cell. Competent cells of the yeast Y2805 Δgal1 / pYGMF-PB strain were prepared in the same manner as described above.

10 μl of human kidney cDNA library, 100 μl of competent cell of yeast Y2805 Δgal1 / pYGMF-PB strain, 10 μl of Salmon sperm DNA, and 600 μl of PEG / LiAc, were allowed to stand at 30 ° C. for 30 min. Heat-shock was carried out for 15 minutes at ℃. After centrifugation at 3000 rpm for 5 minutes, the cells were suspended in 200 μl of distilled water, plated on YG (YNB & Glucose, UL-) plate, and incubated at 30 ° C. for 2 to 3 days to obtain double transformed colonies.

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

96 wells were used to incubate the colonies under the same conditions, and in the case of a general 96 well plate, the yeast did not grow well, and the culture was cultured in a 96 deep well plate. 600 µl of YPDG (1% yeast extract, 2% Peptone, 1% Dextrose, 1% Galactose) medium was added to the sterilized 96 deep well plate and inoculated with each transformant. At this time, each of the transformants was picked together with other YG (YNB & Glucose, UL-) plates to secure a master cell. The inoculated transformants were placed in a 96 deep well plate incubator (Bioneer Co. Ltd) and incubated for 48 hours at 30 ° C. and 400 rpm while supplying oxygen.

The cells were centrifuged to remove pellets, and then 50 μl each was loaded into a dot blot kit on which NC (Nitrocellulose) membranes were placed, followed by washing twice with 50 μl of 1X TBS. Membrane was added to the sealing bag, Blocking solution (5% skim milk in 1X TBS) and then blocked at room temperature for 2 hours. After removing the blocking solution, the primary antibody (Mouse Anti-PDGF, monoclonal) dissolved in the blocking solution was added at a ratio of 1: 2000 and shake incubation overnight at 4 ° C. After overnight shaking incubation membrane was washed three times for 10 minutes with TTBS (0.05% Tween 20 in 1X TBS) and the secondary antibody (Anti-mouse IgG) at a ratio of 1: 4000 and incubated for 2 hours at room temperature. After washing Membrane with TTBS in the same manner as above, NBT and BCIP were added to the detection buffer, followed by color development in the cow, for qualitative analysis of protein expression and secretion (FIG. 7).

FIG. 7 shows a human kidney-derived cDNA library for yeast Y2805 Δgal1 for primary detection of secretory factor capable of increasing expression of mature PDGF-B (WM) gene in yeast Y2805 Δgal1 / pYGMF-PB using Dot blot technology. It is a result of qualitatively observing the change of PDGF-BB expression amount by transforming / pYGMF-PB. In Fig. 1, 2, and 3 as control, culture supernatant of yeast Y2805 Δgal1 / pYGMF-PB / pACT2-double transformant (double transformed with empty pACT2 without human kidney-derived cDNA) , No. 2 shows the yeast Y2805 Δgal1 / pYGMF-PB culture supernatant, No. 3 shows the yeast Y2805 Δgal1 culture supernatant. Blot did not write the numbers are the numbers displayed in the culture supernatant of yeast Y2805 △ gal1 / a pYGMF-PB are derived from human kidney cDNA conversion double transfected with the introduced pACT2 the transformant (Y2805 △ gal1 / pYGMF-PB / pACT2) Omitted. Samples with higher intensity were selected primarily as compared to the three controls.

Example 4 Quantitative Analysis and Selection of PDGF-BB Expression and Secretion of Double Transformants

ELISA primary analysis was performed on 82 colonies with high PDGF-BB signal in the primary search using dot blot (not shown). The master cells were identified and analyzed by ELISA. ELISA used a kit purchased from Human PDGF-BB DuoSet ELISA Development system (Cat. DY220, R & D system).

The double transformants were inoculated in 5 ml of YPDG (yeast extract 1%, Peptone 2%, Dextrose 1%, Galactose 1%), and then incubated at 30 ° C. and 180 rpm for 48 hours.

For ELISA, firstly, 100 μl of Capture reagent (Recombinant human PDGF Rβ) was diluted in a coating plate and reacted at room temperature overnight. After removing the solution in the well was washed three times for 10 minutes with PBST (0.05% Tween20 in PBS) and then added a blocking solution (1% BSA in PBS) was reacted for 1 hour at room temperature. Then, washed again with PBST and added the same amount of the culture supernatant diluted in a certain multiple with PDGF standard contained in the kit and reacted at room temperature for 2 hours. After washing the wells with PBST, the detection antibody (Goat anti-human PDGF-BB) was added and the reaction was allowed to stand at room temperature for 2 hours. After washing with PBST, Streptavidin-HRP was added thereto. After washing with PBST, Substrate solution (TMB, SIGMA) was added and reacted for 20 minutes at room temperature and in the dark. Then, the stop solution (2N H2SO4) was added and the absorbance at the wavelength of 450 nm and 540 nm was measured. In order to reduce the measurement error, the calibration value obtained by subtracting the absorbance value measured at 540 nm from the value measured at 450 nm was used. In this manner, the ELISA secondary analysis was performed three times with the PDGF-BB standard to obtain an average value of S-numbers to 17 transformants with increased PDGF-BB expression compared to the control. The PDGF-BB concentration in the 17 double transformant culture supernatants is shown graphically in FIG. 8. In FIG. 1, the yeast Y2805 Δgal1 culture supernatant, the second time the yeast Y2805 Δgal1 / YEGα-HIR525 culture supernatant, and the third time the yeast Y2805 Δgal1 / pYGMF-PB / pACT2- culture supernatant.

Example 5 Sequencing of PDGF-BB Secretion Factors

(1) Secretion Factor DNA Amplification of Selected Strains

In the second ELISA, three strains (pS-12, 17, 26) with the best PDGF-BB expression were selected and inoculated in 3 ml of YPD (1% yeast extract, 2% Peptone, 2% Dextrose) to recover DNA. After overnight culture. The cultured cells were centrifuged at 13000 rpm for 10 minutes to remove the supernatant, and then suspended in 50 µl of STES (0.5M NaCl, 0.2M Tris-HCl (pH7.6), 0.01M EDTA, 1% SDS) buffer and centrifuged. . Then, the same amount of STES buffer was added again and glass beads were added thereto and stirred for 5 minutes. 1X TE and RNase A (20mg / ml) were added and reacted at 30 ° C. for 1 hour, followed by Phenol: Chroloform: Isoamyl alcohol = 25: 24: 1 and stirred for 5 minutes. After centrifugation at 13000 rpm for 10 minutes, the supernatant was transferred to a new tube, and precipitation was performed using 3M sodium acetate and 100% EtOH. Then, washed with 70% EtOH, dried and dissolved in distilled water and confirmed the DNA by electrophoresis.

1 μl DNA, primer FSCR: 5'-CTATTCGATGATGAAGATACCCCACCAAAC-3 '(SEQ ID NO: 6) and RSCR: 5'-TGAACTTGCGGGGTTTTTCAGTATCTACGA-3' (SEQ ID NO: 7), 2 μl FSCR (10 pmol), 2 μl RSCR (10 pmol), 5 X After inserting 4µl of PCR premix, the final volume was adjusted to 20µl, followed by 30 cycles of PCR for 1 minute at 95 ° C, 1 minute at 55 ° C, and 3 minutes at 68 ° C, followed by electrophoresis to confirm the insert size. 3 μl of this was used for transformation.

E. coli DH5α competent cells were mixed with the obtained plasmid DNA, electroporation, and cultured in LB and plated on LB plate containing ampicillin to obtain a transformant.

(2) Sequence analysis of PDGF-B secretion factor

The transformants obtained above were randomly selected and inoculated into ampicillin-added LB to recover plasmid DNA, and nucleotide sequences were determined.

After plasmid DNA was isolated and purified from three clones consistently positive among candidates obtained by back-transformation into the YY2805 Δgal1 / pYGMF-PB strain, genes with three different sequences were inserted through sequencing. It was found that it acts as a protein secretion factor (Table 3).

Glutathione S-transferase (GST) contained in pS-12 is well known as a fusion protein used for fusion with a target protein for purification for ease of purification during protein purification. However, no reports or mechanisms have been reported with pS-17 and pS-26 to help secrete and produce PDGF and other proteins.

Example 6: Confirmation of PDGF-BB Productivity Using 5L Fermenter

Among the selected strains, Y2805 Δgal1 / pYGMF-PB / pS-26 was cultured in a high concentration using a 5L fermenter to analyze the possibility of industrial production of PDGF-BB.

The medium composition of the species culture was 2 ~ 20 hr culture with glucose 2%, Yeast Extract 1%, Bactopeptone 2%. The media composition of the culture was 72hr in 2% glucose, 4% Yeast Extract, 1.5% Bactopeptone. The culture was controlled by a fed-batch culture at agitation speed of 500rpm to 900rpm and aeration rate of 2.0vvm (v / v) at 1.0vvm (v / v). New medium (60% glucose, 5% yeast extract) was added after 13 hours of glucose in the medium was almost exhausted, and galactose induction (1.5% galactose of the total volume) was performed after 40 hours of 100 or more when measuring OD _600. . As a result, PDGF-BB could be produced at a maximum concentration of 130ng / ml (FIG. 9).

As described above, by using the optimal secretion factor for PDGF overproduction by the present invention it is possible to mass expression of PDGF-B. Therefore, the present invention is not only possible to reduce the production cost but also very useful for establishing a high efficiency secretion system of various high value protein products.

Attach sequence list electronic file  

Claims (7)

A yeast that produces / secretes PDGF-B with high efficiency, which is co-transformed by platelet-derived growth factor B (PDGF-B) gene and secretory factor gene. The method of claim 1, Yeast for producing / secreting PDGF-B with high efficiency, wherein the PDGF-B gene and secretory factor gene are present in different vectors. The method according to claim 1 or 2, The secretion factor gene is yeast for producing / secreting PDGF-B with high efficiency, which is derived from human kidney. The method according to claim 1 or 2, The secretion factor is cytochrome c oxidase subunit I (CCOS I), characterized in that yeast producing and secreting PDGF-B with high efficiency. The method of claim 4, wherein The vector in which the PDGF-B gene is present is pYGMF-PB having the characteristics shown in FIG. 4, and the vector in which the CCOS I gene is present is pACT2. The method of claim 5, The yeast is a yeast producing and secreting PDGF-B with high efficiency, characterized in that Y2805 Δgal1 / pYGMF-PB / pACT2. (A) separating and cloning platelet-derived growth factor B (PDGF-B) genes and introducing them into expression vectors; (B) transforming the yeast with the expression vector and then culturing to isolate the primary transformed yeast expressing PDGF-B; (C) transforming said primary transformed yeast with a human cDNA library again and isolating secondary transformed yeast from which PDGF-BB is secreted extracellularly; Production method of yeast producing / secreting PDGF-B with high efficiency comprising a.

Images