Abstract:
A putative β-glucosidase gene (bglA), encoding 447 amino acids with a calculated mass of 51.6 kDa, was reported in the complete genome sequence of Bacillus halodurans C-125. This gene sequence was PCR amplified to produce construct pBglA-1. E. coli BL21 (DE3) CodonPlus cells transformed with this construct produced bglA at levels >35 % of the total E. coli cellular proteins, when induced with 0.6 mM IPTG. Different constructs produced with silent mutations in +2 and +3 codons of the gene generally showed similar levels of expression. Approximately 70 % of the expressed enzyme was obtained in the soluble form in E. coli cells transformed with the constructs pBglA-1 to -5, and pBglA-7. However, the construct pBglA-6, which had serine TCA at +3 position instead of native TCG produced this enzyme in an insoluble form. Expression analysis in the early stages after IPTG induction showed that synthesis of bglA was more rapid in the cells transformed with pBglA-6 as compared to other transformants leaving the expressed enzyme with little time to fold and assume native conformation. By lowering the cultivation temperature to 18°C the expression in soluble form, however, could be achieved. This study highlights the significance of synonymous substitutions in the 5′-end coding sequence of bglA gene in determining the rate of expression and folding of the expressed enzyme in E. coli.
Page(s):
153-158
DOI:
DOI not available
Published:
Journal: Pakistan Journal of Biochemistry and Molecular Biology , Volume: 43, Issue: 3, Year: 2010