Design, Construction and Expression of a Synthetic Gene for Metal Binding Proteins

Abstract

Construction of synthetic genes is today the most elegant way to optimize the heterologous expression of a recombinant protein. The availability of sequences of entire genome has significantly increased the number of protein targets which many of them will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. In this study we have optimized a two-step polymerase chain reaction (2-step PCR) method for the fast and extremely accurate synthesis of a 186 bp CUP1 gene encoding yeast Saccharomyces cerevisiae copper metallothionein. A total of the six overlapping oligonucleotides ranged from 43 to 49 in length, designed with the unique restriction sites, were assembled in a single step PCR. The assembly was then further amplified by second PCR to produce a synthetic gene which has been cloned into the pET28a(+) vector to allow the expression of CUP1 gene in E. coli BL21 (DE3) host cell. In order to compare the difference in expression level of the gene with optimized codon usage for E. coli, CUP1gene was redesigned according to codon bias of host cell. A significant increase of expression level of codon optimized gene was obtained compared to original sequence of CUP1 gene of copper metallothionein in yeast Saccharomyces cerevisiae.