Synthesis and expression of a mutanted human basic fibroblast growth actor gene in escherichia coli

摘要

There are high content of E.coli rarely used codons in the nature bFGF gene and a stable secondary structure at the 5'terminal of the gene, that prevented the efficient expression of hbFGF in E.coli. And the natural hbFGF protein is unstable in vitro, while a mutant in which the Cystein residues present at position 78 and 96 are replaced by Serine residues is much more stable and has the same activities as the native protein~(1). So we decided to synthesize a mutant gene which will be more fitful to be expressed in E.coli -- -C78s/C96S hbFGF gene. The sequence of the gene was based on the statistically preferred codons in highly expressed E.coli genes. And the 5'end of the sequence was modified by decreaisng the G+C content without altering the predicted amino acid sequence. And the codons of Cystein at the sites of 78, 96 were replaced by the Serine with hoping to increase the stability of the recombinant products. The whole DNA sequence of the 465 bases was divided into six oligonucleotides of about 80-110 base pairs and with overlaps of 18-21 bases between adjacent oligonucleotides. The overlaps served as primers in the PCR-based gene synthesis of the fragments of the gene. After the PCR reaction the fragments were digested by definite enzyme whose sites had been designed at the end of the respectively oligonucleotides. The three fragments were then ligated together to form the complete gene. After the property of the gene had been confirmed by sequencing the mutant gene was inserted into a vector pBV220. After growth at the permissive temperature, the temperature of the culture was raised to allowing the expression of foreign gene. Cells were then broken by ultrasonic and bFGF was purified by Heparin-sepheroase column chromatography. Proten concentration of recombinant products had the ability to simulate mitogenesis of the NIH 3T3 cells.