Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carrying multiple copies of target gene

Science & Technology Development Journal, 23(2):499-507 Open Access Full Text Article Research Article VNUHCM – University of Science Correspondence Le Kha Han, VNUHCM – University of Science Email: lkhan@hcmus.edu.vn Correspondence Nguyen Tri Nhan, VNUHCM – University of Science Email: ntrnhan@hcmus.edu.vn History  Received: 2020-01-01  Accepted: 2020-04-06  Published: 2020-04-14 DOI : 10.32508/stdj.v23i2.1746 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carryingmultiple copies of target gene Le Kha Han*, Nguyen Cao Kieu Oanh, Nguyen Hieu Nghia, Nguyen Tri Nhan* Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Fibroblast growth factor-2 (FGF-2) is a multifunctional protein that plays an impor- tant role in the regulation of proliferation, differentiation and migration of a variety of cells. The recombinant human FGF-2 (rhFGF-2) is currently used in stem cell culture, medicine and cosmetic products. In this study, we aim to produce secreted rhFGF-2 protein from a Pichia pastoris strain containing multiple copies of the fgf-2 gene to eliminate the disadvantages of intracellular expres- sion systems. Methods: The recombinant Pichia pastoris carrying the fgf-2 gene was cloned by using homologous cloning method. The recombinant strains were screened by PCR reactions us- ing specific primers for the target gene and the AOX1 promoter sequence. Moreover, the copy number of the fgf-2 gene inserted into the P. pastoris genome was identified by semi-quantitative PCR method. The secreted rhFGF-2 protein was collected in the induced BMMY medium at a fi- nal methanol concentration of 0.5%, and purified by one-step heparin affinity chromatography. The quantity and biological activity of the purified protein were determined by competitive ELISA method and MTT assay on NIH-3T3 cell line, respectively. Results: Various recombinant P. pastoris clones carrying different copy numbers of the fgf-2 gene were obtained and categorized into 3 groups: the low copy strains (4-5 copies), medium copy strains (8-11 copies), and high copy strains (more than 20 copies). Among those strains, the 4-copy one produced the rhFGF-2 protein at the highest expression level. After purification, the purity of rhFGF-2 protein reached 98.8%, and the recovery yield was 179.2 mg of protein from 200 mL of flask culture (equivalent to 850 mg/L). The purified rhFGF-2 protein showed similar biological activity on NIH-3T3 cell line with the commercial FGF-2 protein. Conclusion: The recombinant FGF-2 protein was successfully secretory expressed from Pichia pastoris, and successfully purified by only one-step chromatography. Key words: basic fibroblast growth factor, quantification of gene copies, Pichia pastoris, heparin affinity chromatography INTRODUCTION Basic fibroblast growth factor, also known as fibrob- last growth factor-2 (FGF-2), was first discovered in 1973. It plays a crucial role in bone formation1, reg- ulation of tissue repair2–4, wound healing5, and an- giogenesis6. Currently, it is used in stem cell cul- ture, medicine and cosmetics. To enhance the interac- tion between FGF-2 and its receptors, FGF-2 needs to bind to heparin or heparan sulfate proteoglycans7,8. The single chain of this protein contains 146 amino acids (pI = 9.6) and molecular weight is approxi- mately 18 kDa. Specifically, it is easily expressed in prokaryote systems since the post-translational mod- ifications and intra-disulfide bond are not required for its functionality. Up to now, FGF-2 has been ex- pressed in various expression systems as intracellular protein9–12. However, these expression systems are not actually utilized since they have difficulty in dis- rupting the cells and purifying the target protein. The difficulties of intracellular expression systems have been mainly caused by taking time in collecting pro- tein inside the cells and a lot of contaminating pro- teins of the host cells. Most of recombinant FGF-2 protein from intracellular expression systems need to perform 2-step purification in order to reach the ex- pected purity of the desired protein, or tag cleavage step with fusion tag protein9,12. Hence, this study fo- cused on using a secretory protein expression system of P. pastoris in order to overcome these disadvan- tages. The methylotrophic yeast P. pastoris is the most com- mon secretory heterologous recombinant protein ex- pression system. This strain has the ability to secrete target proteins more efficiently than Saccharomyces cerevisiae does, due to a strong promoter, tight reg- ulation, and large size of exosomes13,14. Wild-type P. pastoris X33 strain possesses 2 genes, AOX1 and AOX2, which encode for the methanol metabolism protein. In particular, the AOX1 gene is regulated by the AOX1 promoter, which is a strong promoter. Cite this article : Han L K, Oanh N C K, Nghia N H, Nhan N T. Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carrying multiple copies of target gene. Sci. Tech. Dev. J.; 23(2):499- 507. 499 Science & Technology Development Journal, 23(2):499-507 To enhance the yield of recombinant proteins in P. pastoris, the use of the AOX1 promoter is one of the most common strategies. There are two differ- ent types of homologous recombination-mediated in- sertion, which are (i) ends-in insertion which leads to additive insertion of the target gene and (ii) ends- out insertion which facilitates the replacement of the native AOX1 gene15. The replacement of the AOX1 gene greatly reduces the ability of the host strain to consume methanol, and only one copy of the target gene is inserted into the P. pastoris genome, which can lead to low expression levels of the recombinant protein. Many studies have shown that the existence of multiple copies of recombinant genes can achieve better target protein expression levels16–18. In addi- tion, for this strain, it has been demonstrated that the expression level in optimized fermentation scale of the strain could be higher than shake-flask scale (ap- proximately 16.4-fold) with simple and cheap culture medium19. Thus, P. pastoris is a suitable host cell for industrial scale. Besides, the purification of secreted recombinant protein from P. pastoris might be easier than other systems since most of native secreted pro- teins have a pI lower than 6.020. In a previous study, FGF-2 was successfully expressed and purified in P. pastoris 21. The volumetric productivity of the puri- fied FGF-2 (more than 94% of purity) reached 150 mg/L with optimized shake-flask culture condition and two-step purification; this result was 1.5 times higher than the yield of rhFGF-2 protein expressed in E. coli9. However, this study did not investigate the effect of target gene copy on recombinant protein ex- pression. Therefore, in the present study, we constructed the re- combinant Pichia pastorisX33 carrying multiple copy fgf-2 gene and further investigated the correlation be- tween gene copy number and expression level of ex- tracellular rhFGF-2 protein in Pichia pastoris. With the aimof shortening the downstreamprocess and en- hancing purity, the purification of the rhFGF-2 pro- tein was experimentally performed with a 1-step pu- rification strategy using heparin affinity chromatog- raphy. The biological activity of purified rhFGF-2 was evaluated on the NIH-3T3 cell line. MATERIALS-METHODS Gene, strains and plasmids The gene encoding for FGF-2 was optimized from an origin sequence (UniProtKB - P09038) and then generated by overlap extension PCR method. E. coli host strain DH5a (Thermofisher Scientific, Califor- nia, US) and wild-type P. pastoris X33 (Thermofisher Scientific) were used for cloning and gene expres- sion experiments, respectively. In addition, plas- mid pPICZaA (Thermofisher Scientific) was used for cloning and expression studies. Cloning of pPICZaA/fgf-2 plasmid in E. coli The cDNA encoding for FGF-2 protein was amplified by PCR (Biorad, California, US) with specific primers FG-F and FG-R (primer sequences are shown in Ta- ble ?? – Supplementary document). The cDNA was cloned into the pPICZaA plasmid under the con- trol of the AOX1 promoter by eClone-homologous cloning kit (Laboratory of Molecular Biotechnology, Viet NamNational University – University of Science, Viet Nam). The pPICZaA/fgf-2 plasmid was trans- ferred into E. coli DH5a and incubated in LB-Zeocin medium (Himedia, Mumbai, India) overnight. The transformants were screened by PCR using fgf-2 gene specific primers. The plasmid of randomly chosen positive colonies was extracted, purified by alkaline- SDS lysis method, and verified by PCR using the primers AOX-F/FG-R and primers AOX-F/AOX-R (primer sequences are shown in Table ?? – Supple- mentary document). Afterwards, the recombinant vectors pPICZaA/fgf-2 were confirmed for the cor- rect open reading frame by AOX1 promoter sequence analysis. Cloning of pPICZaA/fgf-2 plasmid in P. pas- toris In the present study, P. pastoris X33:: Mut+ wild type was used for cloning. The plasmid of positive E. coli colonies was extracted by alkaline-SDS lysis method, then transferred into P. pastoris X33 by electropora- tion (Biorad, California, US). Plasmid pPICZaA/fgf- 2 was integrated into host genome through recom- binant homologous site on the AOX1 promoter se- quence. To improve the recombinant homologous yield, the plasmid pPICZaA/fgf-2 was linearized by SacI enzyme (Thermo Fisher Scientific). The transfor- mants were selected on YPD-zeocin medium (Hime- dia) and further identified by PCR using the primer set of FG-F and FG-R. To investigate insertion type, the genome of recombinant P. pastoris was extracted andPCRwas performedwith the primer set ofAOX-F and AOX-R. Indirect quantification of recombinant fgf- 2 gene copy number in P. pastoris The genome of recombinant P. pastoris strains was ex- tracted to perform PCR.The relative quantification of recombinant fgf-2 gene copy number was indirectly 500 Science & Technology Development Journal, 23(2):499-507 quantified by PCR products and compared with the standard curve. Wild-type P. pastorisX33:: Mut+ was used as a genome concentration standard. The num- ber of gene copies was calculated by the ratio of the brightness of target gene band (Y) and AOX1 gene band (X) (Figure 1). The results of PCRwere analyzed by electrophoresis and the brightness of band was de- termined by ImageJ software. Shake-flask expression in P. pastoris A colony was picked up from the plate and overnight pre-cultured in 10 mL YPD-zeocin medium at 300C and 250 rpm. Then, the culture was transferred into 10 mL BMGY medium (Himedia) with a ratio of 1:10 (v/v). When OD600 reached 2.0 – 6.0, the cells were harvested by centrifugation at 5000 rpm (40C for 5 minutes), and the pellet collected. After that, the pellet was resuspended in 30 mL BMMY medium. In order to induce the expression of the fgf- 2 gene, methanol (Xilong Scientific, Shantou, China) was added every 24 hours to a final concentration of 0.5% (v/v). After 72 hours, the supernatant of the culture was collected by centrifugation at 5000 rpm, 40C in 30 minutes. The rhFGF-2 expression was de- termined by SDS-PAGE gel (Himedia) using silver staining (Xilong Scientific) and competitive ELISA (Thermo Fisher Scientific). Purification of rhFGF-2 by heparin-affinity chromatography Considering the isoelectric point and specific affinity of the target protein, heparin-affinity chromatogra- phy was chosen to purify rhFGF-2. The HiTrap Hep- arin HP 5mL column (GE Healthcare, Chicago, US) was equilibrated with 25 mL Buffer A (20 mM Tris- HCl (Biobasic, Toronto, Canada), pH 7.5) at a rate of 5 mL/min. Subsequently, the supernatant was applied to the column at a rate of 5 mL/min. After that, the columnwas washed with 50mL Buffer A.The rhFGF- 2 proteinwas eluted by stepwisemethodwith a variety of Buffer B (20mMTris-HCl, pH7.5, 2MNaCl (Schar- lau, Barcelona, Spain)) concentration. The purity and yield of purification were determined by SDS-PAGE using silver staining, ELISA, and Gel Analyzer 2010a software. Qualification of rhFGF-2 protein by SDS- PAGE andWestern blotting SDS-PAGE method was performed using a 15% gel according to the method of Laemmli. To detect the target protein expression, proteins in the gel were ran with low range protein marker (GE Health Care) and stained with 0.03% silver nitrate. Western blotting was performed to verify the pres- ence of the rhFGF-2 protein. The in-gel protein af- ter running SDS-PAGE was transferred onto nitro- cellulose membrane (GE Health Care) and probed with mouse anti-human FGF-2 IgG (Sigma-Aldrich, Missouri, USA). The anti-mouse horseradish perox- idase (Sigma-Aldrich) was used as a secondary an- tibody. Detection of rhFGF-2 was carried out using Supersignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and ImageQuant LAS 500 (GE Health Care). Quantification of rhFGF-2 protein by com- petitive ELISA The expression level of recombinant protein wasmea- sured by competitive ELISA method. After centrifu- gation, the supernatant of culture was collected. Be- fore performing ELISA, the supernatant was diluted to lower protein concentration, depending on the pro- tein concentration range of the standard curve, which was built with the commercialized rhFGF-2 (cata- log number: 233-FB-025, R&D system, Minneapolis, US) as the standard. The supernatant was immobi- lized to the wells of a 96-well plate overnight. Un- bound protein was washed 3 times with PBS-T and blanks on the well surface were filled with 2% BSA (Sigma-Aldrich) incubated for an hour. Mouse anti- human FGF-2 IgG (Sigma-Aldrich) and anti-mouse horseradish peroxidase (Sigma-Aldrich) were used as primary and secondary antibodies, respectively. Af- terwards, an addition of TMB (Thermo Fisher Scien- tific) was performed to react with horseradish peroxi- dase. This reaction was stopped by HCl 2N (Scharlau, Barcelona, Spain). The concentration of rhFGF-2 was determined through the absorbance of the mixture at 450 nm by Multiskan Ascent (Thermo Fisher Scien- tific) and via the standard curve. Analysis of biological activity of rhFGF-2 The bioactivity of FGF-2 was evaluated by its abil- ity to stimulate the proliferation of NIH-3T3 cell line (ATCC), which has a number of FGF receptors on its cell surface. Based on NIH-3T3 cell numbers in cul- ture medium, bioactivity of rhFGF-2 was compared with commercialized FGF-2 (catalog number: 233- FB-025, R&D system, Minneapolis, US) and negative control (without FGF-2 andwithout cell). The cell was cultured inDMEM/F12without FBS (Sigma-Aldrich) and incubated with target protein for 36 hours. Af- ter this growth period, the cells were incubated with MTT solution (Sigma-Aldrich) for 4 hours to form in- soluble formazan dye. After solubilization, the for- mazan dye was quantitated using Multiskan Ascent 501 Science & Technology Development Journal, 23(2):499-507 Figure 1: Illustration of indirect quantitative gene copy method by PCR and ImageJ. X, intensity of AOX1 gene band; Y, intensity of FGF-2 band. Figure 2: Construction of recombinant E. coli DH5a carrying pPICZaA/fgf-2 plasmid. A, Schematic diagram of forward and reverse amplification primer sets; B, Result of PCR using primer set of FG-R and AOX-F, Lane 1: DNA ladder, Lane 2: Negative Control, Lane 3-7: 5 suspected colonies; C, Result of PCR using primer set of AOX-R and AOX-F, Lane 1: DNA ladder, Lane 2: Negative control, Lane 3: pPICaA plasmid (513 bp), Lane 4-8: suspected pPICZaA/fgf-2 plasmids. 502 Science & Technology Development Journal, 23(2):499-507 software. The measured absorbance directly corre- lates to the number of viable cells. This experiment was performed in triplicate, and the results were sta- tistically analyzed by Graphpad Prism 6.0 software (GraphPad, Inc., CA, USA). The laboratory unit of proliferative bioactivity was calculated as follows: LU  unit ng  = 1x10 6 ED50( mgml ) RESULTS Construction of pPICZaA/fgf-2 plasmid The gene encoding for FGF-2 was cloned into pPICZaA plasmid by eClone kit based on homolo- gous recombinant mechanism. The recombinant vec- tor was transferred into E. coli DH5a . The success- fully transformed host cells were selected based upon their ability to grow on the LB medium containing zeocin, followed by colonyPCR to determine the pres- ence of the fgf-2 insert. The plasmid was extracted from the positive clones and further confirmed for the presence of the fgf-2 gene; the direction of the fgf- 2 gene in the plasmid was confirmed by PCR with 2 different primer sets (Figure 2A). The PCR with the primer set of AOX5’-F and FG-R resulted in a single band of expected size for all 5 positive clones (Figure 2B). This implies that the fgf-2 gene was in- serted after the AOX1 promoter and in the same ori- entation with the promoter. The PCR with the primer set of AOX5’-F and AOX3’-R was performed to make sure that only one copy number of fgf-2 gene was in- serted (Figure 2B). Cloning of pPICZaA/fgf-2 plasmid in P. pas- toris In this present study, the wild-type P. pastoris X33 was used for cloning. To enhance the homologous re- combinant yield, pPICZaA/fgf-2 plasmid was cut by SacI enzyme and then transferred into P. pastoris by electroporation. The positive transformants were se- lected through their ability to grow on YPD-zeocin medium and further subjected to colony PCR with fgf-2 primers in order to verify the presence of the pPICZaA/fgf-2 plasmid. Thewildtype P. pastorisX33 and the pPICZaA/fgf-2 plasmid were used as nega- tive and positive control, respectively. The PCR re- sults showed that there was a single 486 bp band cor- responding to a unique band of the positive control (Figure 3A). It indicated that P. pastoris carrying fgf- 2 gene was successfully cloned. Afterwards, the genotype of these transformants were determined by PCR with the primer set of AOX5’-F and AOX3’-R. The PCR results of all transformants showed that there were two bands at 2.2 kbp and 983 bp, equivalent to the length of AOX1 gene and fgf- 2 gene with their own promoter, respectively. It re- vealed that all recombinant strains had additive in- sertion integration. Therefore, all strains were further determined for the fgf-2 gene copy number of each strain (Figure 3B). As the result of fgf-2 gene copy number quantification (Figure 3C), we succeeded in cloning multiple gene copy number of P. pastoris. Comparison of protein expression among different fgf-2 gene copy strains All strains were classified into three groups based on the copy number of fgf-2 gene: low copy strains (4-5 copies), medium copy strains (8-11 copies), and high copy strains (more than 20 copies). To investigate the effect of gene copy number variations on rhFGF- 2 protein expression, one transformant of each group was cultivated in 100 mL BMMY medium and in- duced by 0.5% methanol. The gene copy number of each group was as follows: 4 (low), 11 (medium), and 22 (high) copies. Every 24 hours, the biomass was col- lected to measure OD600nm and the supernatant was used to analyze rhFGF-2 expression by ELISA and SDS-PAGE. The results showed that all three strains had a commonpattern of the growth curve (Figure 4). The highest OD600nm reached 10-12 after 48 hours of cultivation. It indicated that the integrated gene copy number was not affected by the growth of this strain. Nevertheless, the expression levels among the 3 strains were considerably different (Figure 4). As the result, the expression level of the 4-copy strain in- creased slightly from 24 hours to the end of culture time. In contrast, the expression level of the 11-copy and the 22-copy strains dramatically decreased after 24 hours, despite the fact that growth was changed minimally. Moreover, the lowest copy strain reached a higher rhFGF-2 protein production than the others at 24, 48 and 72 hours, respectively. Consequently, this strain was selected to produce the rhFGF-2 pro- tein, which later on would be studied further for the purification process and bioactivity assay. Purification and identification of rhFGF-2 The P. pastoris X33::fgf-2 was cultivated in 200 mL BMMY at 300C, 250 rpm and induced by 0.5% methanol. After 72 hours, the culture supernatant was collected in order to purify the rhFGF-2 protein by heparin affinity chromatography. The target pro- tein was eluted by using a gradient from 0 to 100% of Buffer B. The protein purity and yield of purification were determined by SDS-PAGE and ELISA, respec- tively. 503 Science & Technology Development Journal, 23(2):499-507 Figure 3: Cloning of pPICZaA/fgf-2 plasmid into P. pastoris. A. Colony PCR results using FG-F/FG-R primers of transformants which grew on YPD-zeocine, Lane 1: DNA ladder, Lane 2: Negative control, Lane 3: P. pastoris X33, Lane 4, pPICZaA/fgf-2 plasmid as positive control, Lane 5-14: transformants. B, PCR results using AOX-F/AOX- R primers of positive transformants which carries pPICZaA/fgf-2 plasmid, Lane 1: DNA ladder, Lane 2: Negative control, Lane 3: P. pastoris X33, Lane 4: pPICZaA/fgf-2 plasmid, Lane 5-14: positive transformants. C, Specific fgf-2 gene copy number of all positive transformants. As seen from the SDS-PAGE results, there were two bands between 14.4 kDa and 20.1 kDa in the 60% - 90%B eluted fraction (well 4-6, Figure 5). These frac- tions were further confirmed by Western blotting by specific antibody (well 9-11, Figure 5) and revealed that both bands were the rhFGF-2 protein. The up- per band was predicted to be the glycosylated rhFGF- 2 protein. The concentration of purified rhFGF-2 protein was qualified by ELISA based on the standard curve, and the purity was determined by Gel Analyzer software. The results showed that rhFGF-2 protein after purifi- cation reached 179.2 mg per 200 mL culture (equiva- lent to 850 mg/L) and the purity reached 98.8%. The recovery was estimated as approximately 6.13%. In conclusion, the highly purified rhFGF-2 protein obtained from P. pastoris X33::fgf-2 could accelerate the proliferation of mouse fibroblasts, equivalent to commercialized product, with laboratory units reach- ing 5.6 – 8.94 x 104 unit/ng. DISCUSSION In this study, the low copy number of target gene re- sulted in the highest expression level which was sim- ilar to previous studies of multi-copy strains, even though the growth of the host strain was not affected by the integrated gene copy number. Many studies have demonstrated that multiple copy number of in- tegrated gene could improve the expression level of the target protein. However, when the optimum is reached, further increase can sometimes cause neg- 504 Science & Technology Development Journal, 23(2):499-507 Figure 4: The growth and FGF-2 expression level of various fgf-2 gene copy number strains was evaluated by OD600 and ELISA. OD600 (line with dots) andOD450 (bar) were graphed as circle ( ) and dark grey – 4 copies, rectangle() andmedium grey – 11 copies, triangle (4) and light grey – 22 copies, respectively. ELISA experiment was performed in triplicate. ative effects 22,23. For instance, high copy P. pastoris strain might suffer from protein folding-related ox- idative stress and insufficient supply of carbon and en- ergy sources24. As a result, the yield of FGF-2 protein in this study was lower than other expression systems in previous studies of P. pastoris9,21, where the culture condition was optimized for methanol concentration, pH, and induction time. Therefore, the efficiency of the P. pas- toris strain in this study could be improved by op- timized shake-flask condition21 and be further en- hanced in up-scale production, if dissolved oxygen, pH, temperature and carbon source feeding strategy could be controlled19,25. In short, P. pastorismight be a promising strain for high yield production of FGF-2 protein. On the other side, the ease of purification in P. pastoris with specific affinity chromatography might bring more benefits than other intracellular expression sys- tems9,26, due to lower content of extracellular pro- teins secreted from the host cells. As compared with another study of FGF-2 expressed from P. pastoris21, the purification method of our study was better at re- ducing time and costs for downstream processes since we only performed one-step. The differences in re- sults might be caused by the types of chromatogra- phy. Moreover, the recovery of purification in this study (6.13%) was higher than in a previous study with same type of chromatography (4.49%) 10. This might be affected by differences of equilibration and washing buffer. Briefly, heparin affinity chromatogra- phy is a rationale choice with good potential for short- ening the purification process. After purification, two bands of FGF-2 protein were detected by Western blotting. The original FGF- 2 protein is a non-glycoprotein; nevertheless, nat- ural glycosylation processing can occur at hydroxy groups of threonine and/or serine residues in pro- tein inside yeast cells27. These amino acids were ap- proximately 11.6% of the total peptide sequence of rhFGF-2 protein. Hence, we predicted that the upper band is glycosylated FGF-2 protein. The bioactivity of the mixture of glycosylated FGF-2 protein and non- glycosylated FGF-2 protein was demonstrated to be equivalent to commercialized FGF-2 protein, based on its ability to stimulate the proliferation of the NIH- 3T3 cell line. Therefore, glycosylation did not affect the bioactivity of the FGF-2 protein. CONCLUSIONS In short, our study was successful in cloning multiple copy of fgf-2 gene into Pichia pastoris strain. In com- parison with other multiple copy fgf-2 gene strains, the 4-copy fgf-2 gene strain has the ability to express 505 Science & Technology Development Journal, 23(2):499-507 Figure 5: FGF-2 protein purification by heparin affinity chromatography using stepwise method. Lane 1: Protein ladder, Lane 2-6: SDS-PAGE result of eluted fraction at 50%, 60%, 70%, 80% and 90% of Buffer B, respec- tively, Lane7-11: WesternBlotting result of eluted fraction at 50%, 60%, 70%, 80%and90%of Buffer B, respectively. Table 1: Bioactivity comparison of rhFGF-2 from P. pastoris and commercialized FGF-2 Criteria FGF-2 from P. pastoris Commercialized FGF-2 p-value ED50 (ng/ml) 14.52 3.34 20.80 3.60 0.6619 Hill Slope 1.202 0.3196 1.689 0.415 0.0566 R2 0.9048 0.9577 - LU (unit/mg) 5.6 – 8.94 x 104 4.10 – 5.81 x 104 - Experimental value was represented as Mean SEM. the highest level of rhFGF-2 protein. The concentra- tion of purified rhFGF-2 reached 179.2 mg per 200mL of shake-flask culture (equivalent to 850 mg/L), with 98.8% purity after 1-step heparin affinity chromatog- raphy. In addition, the rhFGF-2 protein showed simi- lar biological activity on theNIH/3T3 cell line as com- mercialized FGF-2 protein. LIST OF ABBREVIATIONS USED AOX1: Aldehyde Oxidase 1 BMMY: Buffer Methanol-Complex Medium bpP: Base pair E. coli: Escherichia coli ELISA: Enzyme-linked immunosorbent assay fgf-2: Fibroblast Growth Factor-2 kDa: Kilo Dalton LB: Luria Broth OD: Optical Density PAGE: Polyacrylamide Gel Electrophoresis P. pastoris: Pichia pastoris PCR: Polymerase Chain Reaction SDS: Sodium Dodecyl Sulfate SDS-PAGE: Sodium Dodecyl Sulfate – Polyacry- lamide Gel Electrophoresis YPD: Yeast extract – Peptone – Dextrose medium ACKNOWLEDGEMENTS This work was supported by VNUHCM - Univer- sity of Science research project funding, Laboratory of Molecular Biotechnology and Department of Molec- ular and Environmental Biotechnology infrastructure of VNUHCM - University of Science. AUTHOR’S CONTRIBUTION This study was designed by Nguyen Hieu Nghia. Nguyen Cao Kieu Oanh contributed on data collec- tion. Data analysis and interpretation for the work were carried out by Nguyen Hieu Nghia, Nguyen Cao KieuOanh andLeKhaHan. NguyenTriNhandrafted the article and Le Kha Han wrote it. The article was critically revised and approved to be published by Nguyen Tri Nhan. 506 Science & Technology Development Journal, 23(2):499-507 CONFLICTS OF INTEREST All author declare that they have no conflicts of inter- est. REFERENCES 1. Nagai H, Tsukuda R, Mayahara HJB. Effects of basic fibrob- last growth factor (bFGF) on bone formation in growing rats. 1995;16(3):367–373. Available from: https://doi.org/10.1016/ 8756-3282(94)00049-2. 2. Rabchevsky A, Fugaccia I, Turner A, Blades D, Mattson M, n Scheff SJE. Basic fibroblast growth factor (bFGF) enhances functional recovery following severe spinal cord injury to the rat. 2000;164(2):280–291. PMID: 10915567. Available from: https://doi.org/10.1006/exnr.2000.7399. 3. Park CM, b Hollenberg MJJD. Basic fibroblast growth fac- tor induces retinal regeneration in vivo. 1989;134(1):201–205. Available from: https://doi.org/10.1016/0012-1606(89)90089- 4. 4. Murakami S, Takayama S, Ikezawa K, Sltimabukuro Y, Kita- mura M, Nozaki T, et al. Regeneration of periodontal tissues by basic fibroblast growth factor. 1999;34(7):425–430. Avail- able from: https://doi.org/10.1111/j.1600-0765.1999.tb02277. xPMid:10685372. 5. McGeeGS, Davidson JM, Buckley A, SommerA,Woodward SC, Aquino AM, et al. Recombinant basic fibroblast growth fac- tor accelerates wound healing. 1988;45(1):145–153. Available from: https://doi.org/10.1016/0022-4804(88)90034-0. 6. Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz DJN. Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. 1987;325(6101):257. Available from: https://doi.org/ 10.1038/325257a0PMid:2433585. 7. Faham S, Hileman R, Fromm J, Linhardt R, Rees DJS. Heparin structure and interactions with basic fibroblast growth factor. 1996;271(5252):1116–1120. PMID: 8599088. Available from: https://doi.org/10.1126/science.271.5252.1116. 8. Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DMJC. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. 1991;64(4):1–8. Available from: https://doi.org/10.1016/0092- 8674(91)90512-W. 9. Gasparian M, Elistratov P, Drize N, Nifontova I, Dolgikh D, Kirpichnikov MJB. Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2). 2009;74(2):221–225. PMID: 19267679. Available from: https: //doi.org/10.1134/S000629790902014X. 10. An N, Ou J, Jiang D, Zhang L, Liu J, Fu K, et al. Expres- sion of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds. 2013;14(2):3556– 3567. PMID: 23434658. Available from: https://doi.org/10. 3390/ijms14023556. 11. Wu X, Kamei K, Sato H, i Sato S, Takano R, IchidaM, et al. High- level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus. 2001;21(1):192–200. PMID: 11162406. Available from: https://doi.org/10.1006/prep.2000. 1358. 12. Soleyman MR, Khalili M, Khansarinejad B, Baazm MJIRCMJ. High-level expression and purification of active human FGF-2 in Escherichia coli by codon and culture condition optimiza- tion. 2016;18(2). PMID: 27175305. Available from: https: //doi.org/10.5812/ircmj.21615. 13. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. Fems microbiology reviews. 2000;24(1):45–66. PMID: 10640598. Available from: https://doi.org/10.1111/j.1574-6976.2000.tb00532.x. 14. AhmadM, Hirz M, Pichler H, Schwab HJ. Protein expression in Pichia pastoris: recent achievements andperspectives for het- erologous protein production. 2014;98(12):5301–5317. PMID: 24743983. Available from: https://doi.org/10.1007/s00253- 014-5732-5. 15. Klinner U, Schäfer B. Genetic aspects of targeted insertion mutagenesis in yeasts. 2004;28(2):201–223. PMID: 15109785. Available from: https://doi.org/10.1016/j.femsre.2003.10.002. 16. Nordén K, Agemark M, Danielson JÅ, Alexandersson E, Kjell- bom PM, Johanson UJ. Increasing gene dosage greatly en- hances recombinant expression of aquaporins in Pichia pas- toris. 2011;11(1):47. PMID: 21569231. Available from: https: //doi.org/10.1186/1472-6750-11-47. 17. Athmaram T, Saraswat S, Singh AK, Rao MK, Gopalan N, Suryanarayana V, et al. Influence of copy number on the expression levels of pandemic influenza hemagglutinin re- combinant protein in methylotrophic yeast Pichia pastoris. 2012;45(3):440–451. PMID: 22940846. Available from: https: //doi.org/10.1007/s11262-012-0809-7. 18. Mansur M, Cabello C, Hernández L, País J, Varas L, Valdés J, et al. Multiple gene copy number enhances insulin pre- cursor secretion in the yeast Pichia pastoris. 2005;27(5):339– 345. PMID: 15834796. Available from: https://doi.org/10.1007/ s10529-005-1007-7. 19. Markošová K, Weignerova L, Rosenberg M, Křen V, Rebroš M. Upscale of recombinant a-L-rhamnosidase production by Pichia pastoris MutS strain. 2015;6(1140). PMID: 26539173. Available from: https://doi.org/10.3389/fmicb.2015.01140. 20. Huang CJ, Damasceno LM, Anderson KA, Zhang S, Old LJ, Batt CA, et al. A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. 2011;90(1):235–247. PMID: 21305280. Available from: https://doi.org/10.1007/s00253- 011-3118-5. 21. Mu X, Kong N, Chen W, Zhang T, Shen M, Yan W. High-level expression, purification, and characterization of recombinant human basic fibroblast growth factor in Pichia pastoris. Pro- tein Expression and Purification. 2008;59(2):282–288. PMID: 18378165. Available from: https://doi.org/10.1016/j.pep.2008. 02.009. 22. Abad S, Kitz K, HörmannA, Schreiner U, Hartner FS, Glieder AJ. Real-time PCR-based determination of gene copy numbers in Pichia pastoris. 2010;5(4):413–420. PMID: 20349461. Available from: https://doi.org/10.1002/biot.200900233. 23. Zhu T, Guo M, Tang Z, Zhang M, Zhuang Y, Chu J, et al. Effi- cientgenerationofmulti-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pas- toris. 2009;107(3):954–963. PMID: 19486418. Available from: https://doi.org/10.1111/j.1365-2672.2009.04279.x. 24. Zhu T, Guo M, Zhuang Y, Chu J, Zhang SJ. Understand- ing the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes. 2011;89(4):1127–1135. PMID: 20981418. Available from: https://doi.org/10.1007/s00253-010-2944-1. 25. Markošová K, Camattari A, Rosenberg M, Glieder A, Turner NJ, Rebroš MJ. Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris. 2018;40(1):127– 133. PMID: 29019030. Available from: https://doi.org/10.1007/ s10529-017-2450-y. 26. Sheng Z, Chang SB, Chirico WJ. Expression and purification of a biologically active basic fibroblast growth factor fusion protein. Protein Expression and Purification. 2003;27(2):267– 271. Available from: https://doi.org/10.1016/S1046-5928(02) 00601-0. 27. Gemmill TR, Trimble RB. Overview of N-and O-linked oligosaccharide structures found in various yeast species. 1999;1426(2):227–237. Available from: https://doi.org/10. 1016/S0304-4165(98)00126-3. 507