2019 ; Vol.29-2: 274~282
|Author||Aatif Amin, Arslan Sarwar, Mushtaq A Saleem, Zakia Latif, Stanley J Opella|
|Place of duty||Department of Microbiology and Molecular Genetics, University of the Punjab, Pakistan|
|Title||Expression and Purification of Transmembrane Protein MerE from Mercury-Resistant Bacillus cereus|
J. Microbiol. Biotechnol.2019 ;
|Abstract||Mercury-resistant (HgR) bacteria were isolated from heavy metal polluted wastewater and soil
collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3
showed resistance up to 40 μg/ml against mercuric chloride (HgCl2). 16S rDNA ribotyping
and phylogenetic analysis were performed for the characterization of selected isolates as
Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3
(KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-
100% homology with the corresponding region of the merA gene of already reported mercuryresistant
Gram-positive bacteria. The merE gene involved in the transportation of elemental
mercury (Hg0) via cell membrane was cloned for the first time into pHLV vector and
transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE)
was expressed and the native MerE protein was obtained after thrombin cleavage by size
exclusion chromatography (SEC). The purification of fusion/recombinant and native protein
MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC)
involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing
30 μg/ml of HgCl2 was designed to check the detoxification ability of selected strains. It
resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76%
detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).|
|Key_word||16S rRNA, Ni-NTA chromatography, pHLMerE, thrombin, Hg-detoxification|
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