The Optimization Of The Structure And The Function Of HbFGF Expressed In E.coli

Objectives: In our previous study, to improve the expression efficiency of human basic Fibroblast Growth Factor(hbFGF) expressed in Escherichia coli, we lowered the G+C content in hbFGF's TIR region and used more preferred codons by E.coli. Meanwhile we found the low insoluble product with a large quantity of dimer andhigher multimer in the protein solution. It greatly decrease the bioactivity of hbFGF. In this study, we set out to optimize the solubility and bioactivity of hbFGF by aa mutation.Methods: According to the research of Song et al, the free energy of the Translation Initiation Region(TIR) and the Codons preference in the same region were adjusted. We concentrated our adjustment on the sequence encoding the first 20 aa downtstream the initiation codon ATG, the software DNASIS v2.5 were used for the calculation of the free energy. There are 4 cysteins in the protein hbFGF, cysteins 78 and 96 are localized on the surface of hbFGF,whereas cystein 34 is completely buried and cystein 101 is partly buried within the folded peptide chain. So the Cys78 and Cys96 are especially prone to intramolecular disulphide-bond formation. We converted these two cysteins into serines. Result:1. We obtained the full length gene of hbFGF coding sequence with PCR, and adjusted the G+C content according the software DNASISv2.5, and replaced the Cys78 and Cys96 with Serines by site-directed mutagenesis.2. Sequence result suggested one of the recombinant is correctly synthezied and cloned.3. The higher expression of 16% are obtained, and the dimers formation were reduced to below 5%, and inclusion body come down too.4. The mutant protein were found to possess higher bioactivity than native standard protein.Conclusion:It's possible to improve the expression efficiency by adjusting the G+C content of TIR region and codon usage. Replacement the cystein 78 and cystein 96 into serines, higher bioactivity gained compared with control hbFGF protein, and decreased amout of dimer and inclusion body, this suggested that free thiol group of Cys78 and Cys96 is the main reason of dimer and multimer formation.