SHuffle™ T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties

Abstract Background Human fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1. Methods A synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts ( BL21 (DE3) , Rosetta-gami™ 2 (DE3), SHuffle™ T7 ). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties. Results The highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500 mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84 ng/mL. Conclusion Although SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.