Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

Abstract

Introduction: Human basic fibroblast growth factor (hbFGF) is a highly valuable multifunctional protein that plays a crucial role in various biological processes. In this study, we aim to accomplish the scaling-up production of mature hbFGF (146aa) by implementing a high cell-density fermentation and purification process on a 500-L scale, thereby satisfying the escalating demands for both experimental research and clinical applications. Methods: The hbFGF DNA fragment was cloned into a mpET-3c vector containing a kanamycin resistance gene and then inserted into Escherichia coli BL21 (DE3) plysS strain. To optimize the yield of hbFGF protein, various fermentation parameters were systematically optimized using BOX-Behnken design and further validated in large-scale fermentation (500-L). Additionally, a three-step purification protocol involving CM-Sepharose, heparin affinity, and SP-Sepharose column chromatography was developed to separate and purify the hbFGF protein. Isoelectric focusing electrophoresis, MALDI-TOF/MS analysis, amino acid sequencing, CD spectroscopy, and Western blotting were performed to authenticate its identity. The biological efficacy of purified hbFGF was evaluated using an MTT assay as well as in a diabetic deep second-degree scald model. Results: The engineered strain was successfully constructed, exhibiting high expression of hbFGF and excellent stability. Under the optimized fermentation conditions, an impressive bacterial yield of 46.8 ± 0.3 g/L culture with an expression level of hbFGF reaching 28.2% ± 0.2% was achieved in 500-L scale fermentation. Subsequently, during pilot-scale purification, the final yield of purified hbFGF protein was 114.6 ± 5.9 mg/L culture with RP-HPLC, SEC-HPLC, and SDS-PAGE purity exceeding 98%. The properties of purified hbFGF including its molecular weight, isoelectric point (pI), amino sequence, and secondary structure were found to be consistent with theoretical values. Furthermore, the purified hbFGF exhibited potent mitogenic activity with a specific value of 1.05 ± 0.94 × 106 AU/mg and significantly enhanced wound healing in a deep second-degree scald wound diabetic rat model. Conclusion: This study successfully established a stable and efficient large-scale production process of hbFGF, providing a solid foundation for future industrial production.

Keywords: 500-L fermentation; Escherichia coli BL21(DE3) plysS; hbFGF; optimized production; purification; wound healing.

FIGURE 1

Recombinant plasmid construction and expression of the hbFGF protein. (A) Schematic diagram of the mpET3c-hbFGF recombinant plasmid construction. (B) Kanamycin resistance detection of the mpET3c-hbFGF recombinant plasmid. 1, E. coli BL21 (DE3) plysS-pET3c strain. 2, E. coli BL21 (DE3) plysS-mpET3c/hbFGF engineered strain. (C) Restriction enzyme analysis of the mpET3c-hbFGF recombinant plasmid. Lanes 1 and 4, DNA molecular weight marker. Lane 2, plasmid before digestion. Lane 3, plasmid after digestion. (D) SDS-PAGE (left) and Western blot (right) analyses of the expressed hbFGF protein in E. coli BL21(DE3) plysS. Lanes 1 and 4, non-induced. Lanes 3 and 5, induced by IPTG for 4 h. Lane M, molecular weight marker. Black arrows indicate hbFGF.

FIGURE 2

Optimizing of fermentation parameters of hbFGF E. coli strain in 30 mL of LB medium. (A) The 12-h growth curve of the hbFGF E. coli strain in 30 mL of LB medium at 37°C and 200 rpm. (B–E) The 12-h growth curve of hbFGF E. coli strain under different conditions, including (B) glucose concentrations in the range of 0.5–20 g/L, (C) pH in the range of 6.6–7.4, (D) temperatures of 32°C, 34°C, 36°C, and 38°C, and (E) medium volume (30, 50, 75, and 100 mL). (F) The expression level of hbFGF (f1) and bacterial density (f2) after induction at different OD₆₀₀ values with 0.8 mM IPTG for 1–6 h. (G) The 10-h growth curve of the hbFGF E. coli strain under different inoculations. All experiments were performed in a 250-mL shake flask. Asterisks indicate significant difference (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).

FIGURE 3

Three-dimensional response surface for the bacterial density and hbFGF expression level. (A) Effects on the bacterial density of (a1) temperature and pH; (a2) temperature and IPTG concentration; (a3) temperature and NH₄Cl concentration; (a4) temperature and induction time; (a5) pH and IPTG concentration; (a6) pH and NH₄Cl concentration; (a7) pH and induction time; (a8) IPTG concentration and NH₄Cl concentration; (a9) IPTG concentration and induction time; and (a10) NH₄Cl concentration and induction time. (B) Effect on the hbFGF expression level of (b1) temperature and pH; (b2) temperature and IPTG concentration; (b3) temperature and NH₄Cl concentration; and (b4) temperature and induction time.

FIGURE 4

Process of fermentation of E. coli BL21 (DE3) plysS-mpET3c/hbFGF. (A) Growth curve of hbFGF engineered strain in a 200-L fermentation. (B) SDS-PAGE analysis of the hbFGF expression at the 500-L scale. #1 Lane, non-induced. Lane M, molecular weight marker. (C) Variations in the parameters for hbFGF production at the 500-L scale, including wet cell concentration (OD₆₀₀), temperature, dissolved oxygen (DO) levels, rotation speed, and air ventilation rate. (D) The relative curves of the specific growth rate, pH, glucose addition, and nitrogen source addition with time in a 500-L fermentation. Black arrows indicate hbFGF.

FIGURE 5

Identification and analysis of hbFGF in the purification process. (A) SDS-PAGE analysis of hbFGF after ion exchange and affinity chromatography. Lane 1, supernatant. Lane 2, the flow-through sample from CM-Sepharose. Lane 3, 0.36 M NaCl-eluted sample from CM-Sepharose. Lane 4, 2.0 M NaCl-regenerated sample from CM-Sepharose. Lane 5, flow-through fraction from heparin affinity Sepharose. Lane 6, 2.0 M NaCl-eluted sample from heparin affinity Sepharose. Lane 7, 2.0 M NaCl-regenerated sample from SP-Sepharose. Lane 8, purified hbFGF eluted with 0.5 M NaCl from SP-Sepharose. Lane M, molecular weight marker. (B) RP-HPLC and (C) SEC-HPLC analysis of purified hbFGF. (D) The biological activity of hbFGF on NIH-3T3 cells. (E) Analysis of Isoelectric point, (F) Mass spectrum, (G) molecular peptide mapping coverage, and (H) CD spectrum analysis of purified hbFGF. Black arrows indicate hbFGF.

FIGURE 6

The healing effect of hbFGF on a deep second-degree scald wound model in rats. (A) Photos of the skin wound and wound healing rates of STZ-induced SD rats with deep second-degree scald wounds. (B) Results of Masson (scale bar, 200 μm) and IHC staining (scale bar, 50 μm) and the pathological score of a deep second-degree scald wound model in rats. (C) The expression level of FGF-1, TGF-β1, and hydroxyproline in a deep second-degree scald wound model in rats. Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001, n = 7.