Cloning, expression and purification of mycobacterium tuberculosis RV0309 adhesin protein.

Abstract

Tuberculosis (TB) remains a global burden despite major advances in the design of rapid diagnostics and therapeutics. Mycobacterium tuberculosis (Mtb) adhesin proteins are key in the pathogenicity and virulence of Mtb and are potential biomarkers for diagnostics, drug targets or vaccine candidates. Numerous adhesin proteins have been considered; however, they have not proven to be both highly sensitive and specific in point-of-care tests. The increased emergence of drug-resistant strains, lack of suitable treatment regimens and poor compliance to treatments demonstrate the need for novel targets for therapeutic intervention. Research has shown that the relatively unknown Mtb Rv0309 adhesin membrane protein is essential in the growth, biofilm development, and cellular morphology of Mtb. The Rv0309 gene also enhances mycobacterial intracellular survival after infection, and deletion of the gene may decrease the infecting potential of the resultant mutant strain. Hence, the Rv0309 adhesin encoding L-D transpeptidase was investigated in the current study for its ability to be cloned in glutathione S-transferase (GST)- and polyhistidine- tagged (His-tag) vectors, expressed and purified efficiently for future downstream processing. The Rv0309 gene was amplified using polymerase chain reaction (PCR). The plasmid DNA of pGEX-6P-1 (GST-tagged) and pET28a (His-tagged) vectors was extracted. The Rv0309 DNA and vector DNA were restricted with the appropriate restriction endonucleases, ligated and transformed into E. coli BL21 cells. Transformants were confirmed by colony PCR and plasmid DNA sequencing. Recombinant protein expression was optimised using various isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations, time intervals and visualised using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Protein lysis and purification trials were performed and visualised using SDS-PAGE and Western blotting. The target band found in eluent was excised and sent for peptide mass fingerprinting for protein confirmation. The Rv0309 gene was successfully cloned into both vectors and optimally expressed in Escherichia coli (E. coli). Despite employing an array of lysis and purification techniques, obtaining a pure form of the recombinant protein remained elusive. There was some success with pure protein being obtained during the study; however, the concentration was low and results were not reproducible. The main problems were sub-optimal lysis of the Rec-protein and ineffective binding to the purification column. Based on the bioinformatics analysis performed and information from GenScript, the reason for these problems was the high hydrophobicity of this protein. The insights gained from the lysis, purification and bioinformatic analysis of Rv0309 in this study contribute to the understanding of membrane protein biochemistry and the intricacies associated with Mtb protein purification.