Basic Fibroblast Growth Factor (bFGF) is one of the twenty two members of fibroblast growth factors family with most potent angiogenic and wound healing properties. Though there is an increasing potential application of recombinant bFGF for human use, high cost of peptide in the market, is always a barrier. In this work, a novel application of intein has been explored where intein had proven efficient in increasing the expression of recombinant bFGF when expressed in Escherichia coli. Previously engineered construct pWK3R was modified to form plasmid pWK311ROmpAd, which had two copies of DNA segment encoding a fusion product comprising intein Saccharomyces cerevisiae vascular membrane ATPase (VMA) and bFGF but was devoid of ompA leader sequence. When JM101 [pWK311ROmpAd] was cultivated...[ Read more ]

Basic Fibroblast Growth Factor (bFGF) is one of the twenty two members of fibroblast growth factors family with most potent angiogenic and wound healing properties. Though there is an increasing potential application of recombinant bFGF for human use, high cost of peptide in the market, is always a barrier. In this work, a novel application of intein has been explored where intein had proven efficient in increasing the expression of recombinant bFGF when expressed in Escherichia coli. Previously engineered construct pWK3R was modified to form plasmid pWK311ROmpAd, which had two copies of DNA segment encoding a fusion product comprising intein Saccharomyces cerevisiae vascular membrane ATPase (VMA) and bFGF but was devoid of ompA leader sequence. When JM101 [pWK311ROmpAd] was cultivated using a refined fed-batch fermentation, around 610 mg l^-1 of bFGF was obtained altogether from supernatant and cell lysate. More importantly, the peptide was found to be authentic, soluble and was highly bioactive. Authentic bFGF (abFGF) obtained from cell culture supernatant was purified using salt precipitation, followed by heparin affinity chromatography and dialysis. On the other hand, abFGF found in cell lysate was purified by a different approach using cation exchange chromatography, heparin affinity chromatography and Sephadex G-25 size exclusion chromatography. Due to incompatibility of the peptide with working conditions of commercial biochemical assays, dot blot was implemented for protein quantification in this study. Finally, an overall recovery of 70.2% with 37-fold purification was obtained from supernatant and 61.2% of recovery with 111-fold purification was achieved from cell lysate with these schemes. Purified peptide was also authentic and highly bioactive with ED₅₀ (median effective dose) of 9 ng ml^-1. The platform for expression and purification established in this work was simple and presents an efficient way to produce abFGF for commercial applications in the future.

Keywords: bFGF, E.coli, VMA intein, fed-batch fermentation, salt precipitation, heparin affinity chromatography, dialysis, G-25 size exclusion chromatography, ED₅₀.