Hyper-expression of functional human basic fibroblast growth factor 2 in Escherichia coli

ABSTRACT

The human basic fibroblast growth factor 2 (hbFGF2) is a highly conserved protein that is crucial for cell proliferation, differentiation, and migration. Commercial hbFGF2 is expensive and contributes significantly to the cost of cultivated meat (CM) media, affecting the scalability of the production process. This study aims to reduce media costs by achieving a high yield of purified active hbFGF2 from Escherichia coli T7 Express. A codon-optimized hbFGF2 variant, hbFGF2-G3, was cloned into the pET-28a (+) plasmid and expressed in E. coli T7 Express. Cultivation parameters, including temperature and induction time, were optimized in both shake flasks and batch bioreactors. Optimized conditions yielded a titer of 1.3 g/L in shake flasks at 20 °C and an impressive 2.95 g/L in a batch bioreactor, quantified by densitometry using an in-house purified hbFGF2-G3 as the standard. The protein was subsequently purified using His-trap affinity and size exclusion chromatography. The functional activity of the purified hbFGF2-G3 was validated through FGF/FGFR activation assays in HepG2 cell lines and cell proliferation studies in Ph9F-1x fish cell lines. Our findings demonstrate a cost-effective method for producing active hbFGF2-G3, which has significant implications for the economic viability of CM production.