Molecular Cloning and Expression of Bacterial Mercuric Reductase Gene
AbstractIn order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr) Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and finally mer operon was localized by transforming isolated E. coli plasmid into mercury sensitive (Hgs) host E. coli DH5a cells. Oligonucleotide primers were designed by comparing the known
reported sequences of merA from Gram-negative bacterium (E. coli plasmid R100) and 1695 bp full length merA gene was amplified by PCR. A 1.695-kb DNA fragment of merA was inserted into isopropyl- -D-thiogalactopyranoside (IPTG) inducible bacterial expression vector pQE-30U/A. E. coli DH5 strains harboring the merA constructs showed higher mercury reductase enzyme (MerA) activity and expressed significantly more MerA than the control strains under aerobic conditions. The purified merA gene over expressed in the specific host E. coli BL21(DE3)Plys cells. Finally, expressed MerA protein was purified
by Immobilized Metal-chelate Affinity Chromatography (IMAC) by using Ni- NTA column; and ~66.2 kDa bacterial MerA protein was detected after resolving on 10% sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS PAGE).