High level expression of human basic fibroblast growth factor in <i>Escherichia coli</i>: Evaluating the effect of the GC content and rare codons within the first 13 codons

Abstract

High-level expression of recombinant human basic fibroblast growth factor in Escherichia coli presents research opportunities such as analysis of hbFGF expression after translation initiation region (TIR) mutagenesis. In our study, hbfgf-cDNA was expressed in three stains of E. coli comprising OrigamiB (DE3), BL21 (DE3) and modified strain carrying copies for rare codon tRNAs (BL21 (DE3)-codonplus- RP). During the course of these experiments, we investigated the role of rare codon replacement and of
GC content reduction in N-terminal, just downstream of the ATG start codon. As standardized procedure, two forward primers were designed for modification of N-terminal of hbfgf-cDNA. Nterminally modified genes were PCR amplified and cloned into the expression vector, pET-22b.
Meanwhile, wild-type gene remarkably expressed in all the strains especially in codon plus strain, rare codon substituted hbFGF gene construct surprisingly displayed undetectable levels of protein production; modified gene construct with reduction in GC content of the first 13 codons contributes to 2.5 folds increased expression level. In addition, recombinant hbFGF were purified and the biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of NIH/3T3 cells. Purified rhbFGF exhibited proliferative activity comparable to commercial rhbFGF.