Rare Codon Content of BORIS Affects the Recombinant Proteins Expression in a Codon Bias-Adjusted Escherichia coli Strains

摘要

Objectives: Brother of the regulator of the imprinted site (BORIS) is paralog of multifunctional CCCTC-binding transcription factor (CTCF) that exclusively expressed in male germline cells. BORIS protein consists of three domains, N-terminal (N), 11 zinc fingers (ZF) and C-terminal (C) domain. Its limited known protein-protein interactions require characterisation of this protein. This study aims to produce a set of recombinant truncated BORIS proteins using bacterial expression system.Design: This study was designed to produce BORIS truncated recombinant constructs in the prokaryotic expression system. Firstly, all the gene fragments were amplified by PCR and cloned into the pET16b-SH3 (Cys) vector. The ability of these constructs to induce protein expression was determined via Western blot.Material & Methods: BORIS cDNA was amplified via reverse transcriptase polymerase chain reaction (RT-PCR). The truncated BORIS gene fragments: BORIS-N, BORIS-ZF and BORIS-C were constructed in pGEMT vector. Each domain were cloned into pET16b-SH3 (Cys) and chemically transformed into Escherichia coli (E. coli) BL21 (DE3) pLysS and BL21 Star (DE3) pRARE2. Rare codon analysis was also performed to the gene sequences. The denatured proteins were probed by Western blot using anti-Histidine tag mouse monoclonal antibody.Results: Results showed the successful production of BORIS -N, BORIS-ZF and BORIS-C truncated recombinant constructs in the prokaryotic expression system. The protein expression by Western blots showed BORIS-N, BORIS-ZF and BORIS-C proteins were expressed at 35 kDa, 70 kDa and 18 kDa respectively. BORIS-ZF was failed to express in BL21 (DE3) pLysS. Rare codon analysis showed that the ZF domain of BORIS was predominated with rare codons for arginine.Conclusion: Failure of BORIS-ZF in BL21 (DE3) pLysS could be due to the lack of tRNAs that cause translational error and degradation of protein. Successful expression in BL21 Star (DE3) pRARE2 showed tRNAs compensation toward the rare codons in ZF domain. Hence, BORIS truncated protein expression was optimized by increasing availability of rare tRNA in bacterial host. Production of recombinant truncated BORIS in this study would be significant for the in vitro protein-protein interaction approaches.