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 host strain DH5α (Thermofisher Scientific, California, US) and wild-type X33 (Thermofisher Scientific) were used for cloning and gene expression experiments, respectively. In addition, plasmid pPICZαA (Thermofisher Scientific) was used for cloning and expression studies.

Cloning of pPICZαA/plasmid in

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 false – Supplementary document). The cDNA was cloned into the pPICZαA plasmid under the control of the AOX1 promoter by eClone-homologous cloning kit (Laboratory of Molecular Biotechnology, Viet Nam National University – University of Science, Viet Nam). The pPICZαA/plasmid was transferred into DH5α and incubated in LB-Zeocin medium (Himedia, Mumbai, India) overnight. The transformants were screened by PCR using 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 false – Supplementary document). Afterwards, the recombinant vectors pPICZαA/ were confirmed for the correct open reading frame by AOX1 promoter sequence analysis.

Cloning of pPICZαA/plasmid in

In the present study, X33:: Mut wild type was used for cloning. The plasmid of positive colonies was extracted by alkaline-SDS lysis method, then transferred into X33 by electroporation (Biorad, California, US). Plasmid pPICZαA/ was integrated into host genome through recombinant homologous site on the AOX1 promoter sequence. To improve the recombinant homologous yield, the plasmid pPICZαA/ was linearized by I enzyme (Thermo Fisher Scientific). The transformants were selected on YPD-zeocin medium (Himedia) and further identified by PCR using the primer set of FG-F and FG-R. To investigate insertion type, the genome of recombinant was extracted and PCR was performed with the primer set of AOX-F and AOX-R.

Indirect quantification of recombinant fgf-2 gene copy number in

The genome of recombinant strains was extracted to perform PCR. The relative quantification of recombinant gene copy number was indirectly quantified by PCR products and compared with the standard curve. Wild-type X33:: Mut was used as a genome concentration standard. The number 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 PCR were analyzed by electrophoresis and the brightness of band was determined by ImageJ software.

Shake-flask expression in

A colony was picked up from the plate and overnight pre-cultured in 10 mL YPD-zeocin medium at 30C and 250 rpm. Then, the culture was transferred into 10 mL BMGY medium (Himedia) with a ratio of 1:10 (v/v). When OD reached 2.0 – 6.0, the cells were harvested by centrifugation at 5000 rpm (4C 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 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, 4C in 30 minutes. The rhFGF-2 expression was determined 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 chromatography was chosen to purify rhFGF-2. The HiTrap Heparin 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 column was washed with 50 mL Buffer A. The rhFGF-2 protein was eluted by stepwise method with a variety of Buffer B (20mM Tris-HCl, pH 7.5, 2M NaCl (Scharlau, 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 and Western 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 presence of the rhFGF-2 protein. The in-gel protein after running SDS-PAGE was transferred onto nitrocellulose membrane (GE Health Care) and probed with mouse anti-human FGF-2 IgG (Sigma-Aldrich, Missouri, USA). The anti-mouse horseradish peroxidase (Sigma-Aldrich) was used as a secondary antibody. 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 competitive ELISA

The expression level of recombinant protein was measured by competitive ELISA method. After centrifugation, the supernatant of culture was collected. Before performing ELISA, the supernatant was diluted to lower protein concentration, depending on the protein concentration range of the standard curve, which was built with the commercialized rhFGF-2 (catalog number: 233-FB-025, R&D system, Minneapolis, US) as the standard. The supernatant was immobilized to the wells of a 96-well plate overnight. Unbound 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. Afterwards, an addition of TMB (Thermo Fisher Scientific) was performed to react with horseradish peroxidase. 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 Scientific) and via the standard curve.

Analysis of biological activity of rhFGF-2

The bioactivity of FGF-2 was evaluated by its ability 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 culture 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 and without cell). The cell was cultured in DMEM/F12 without FBS (Sigma-Aldrich) and incubated with target protein for 36 hours. After this growth period, the cells were incubated with MTT solution (Sigma-Aldrich) for 4 hours to form insoluble formazan dye. After solubilization, the formazan dye was quantitated using Multiskan Ascent software. The measured absorbance directly correlates to the number of viable cells. This experiment was performed in triplicate, and the results were statistically analyzed by Graphpad Prism 6.0 software (GraphPad, Inc., CA, USA). The laboratory unit of proliferative bioactivity was calculated as follows:

Construction of pPICZαA/plasmid

The gene encoding for FGF-2 was cloned into pPICZαA plasmid by eClone kit based on homologous recombinant mechanism. The recombinant vector was transferred into DH5α. The successfully transformed host cells were selected based upon their ability to grow on the LB medium containing zeocin, followed by colony PCR to determine the presence of the insert. The plasmid was extracted from the positive clones and further confirmed for the presence of the gene; the direction of the 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 gene was inserted after the AOX1 promoter and in the same orientation 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 gene was inserted (Figure 2B).

Cloning of pPICZαA/plasmid in

In this present study, the wild-type X33 was used for cloning. To enhance the homologous recombinant yield, pPICZαA/plasmidwas cut by I enzyme and then transferred into by electroporation. The positive transformants were selected through their ability to grow on YPD-zeocin medium and further subjected to colony PCR with primers in order to verify the presence of the pPICZαA/plasmid. The wildtype X33 and the pPICZαA/plasmid were used as negative and positive control, respectively. The PCR results showed that there was a single 486 bp band corresponding to a unique band of the positive control (Figure 3A). It indicated that carrying 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 gene with their own promoter, respectively. It revealed that all recombinant strains had additive insertion integration. Therefore, all strains were further determined for the gene copy number of each strain (Figure 3B). As the result of gene copy number quantification (Figure 3C), we succeeded in cloning multiple gene copy number of .

Comparison of protein expression among different gene copy strains

All strains were classified into three groups based on the copy number of 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 induced 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 collected to measure OD and the supernatant was used to analyze rhFGF-2 expression by ELISA and SDS-PAGE. The results showed that all three strains had a common pattern of the growth curve (Figure 4). The highest OD 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 increased 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 protein, which later on would be studied further for the purification process and bioactivity assay.

Purification and identification of rhFGF-2

The X33:: was cultivated in 200 mL BMMY at 30C, 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 protein 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, respectively.

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 fractions 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 upper 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 purification reached 179.2 µg per 200 mL culture (equivalent to 850 µg/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 X33:: could accelerate the proliferation of mouse fibroblasts, equivalent to commercialized product, with laboratory units reaching 5.6 – 8.94 x 10 unit/ng.