Construction of Plasmid Vector for the Expression of LipYΔPE from Mycobacterium tuberculosis H37Rv in Escherichia coli

Abstract

LipY is a gene product from Mycobacterium tuberculosis that plays an important role in the hydrolysis of triacylglycerol (TAG), especially during the dormant stage. LipY is one of the PE family protein which are involved in immune pathogenicity and virulence of mycobacteria. The PE domain of LipY participates in the modulation of LipY activity. LipY protein devoid of its PE-domain (LipYΔPE) exhibits a higher enzymatic activity than LipY. LipYΔPE is also able to elicit a humoral immune response and might represent a potent immune dominant antigen. In this study, a plasmid vector for the expression of LipYΔPE from M. tuberculosis H37Rv in E. coli was constructed. In order to purify the LipYΔPE, a DNA segment encoding His6-tag was added at the 3’-end. This study aims to obtain a more efficient LipYΔPE expression from M. tuberculosis H37Rv in E. coli by using pRHA vector. LipYΔPE was first inserted into a TOPO-cloning vector, double-digested with NdeI and XhoI, and ligated into the pRHA expression vector. The successful integration of the lipYΔPE gene into the pRHA vector was verified with PCR colony screening, resulting in 882bp on gel electrophoresis. Double digestion with NdeI and XhoI visualized on gel electrophoresis showed two fragments: pRHA vector and lipYΔPE gene with the size 3.9 kb and 882 bps respectively. The DNA sequencing analysis showed lipYΔPE-His6 gene in correct orientation sequence. Overall, this study has successfully cloned the gene encoding lipYΔPE- His6 with size of 882bp, corresponding to 294 amino acids.