Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression and Purification of Transmembrane Protein MerE from Mercury-Resistant Bacillus cereus

  • Amin, Aatif (Department of Microbiology and Molecular Genetics, University of the Punjab) ;
  • Sarwar, Arslan (Department of Microbiology, Faculty of Life Sciences, University of Central Punjab) ;
  • Saleem, Mushtaq A. (Department of Microbiology, Faculty of Life Sciences, University of Central Punjab) ;
  • Latif, Zakia (Department of Microbiology and Molecular Genetics, University of the Punjab) ;
  • Opella, Stanley J. (Department of Chemistry and Biochemistry, University of California)
  • Received : 2017.04.28
  • Accepted : 2017.08.04
  • Published : 2019.02.28
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Abstract

Mercury-resistant ($Hg^R$) 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 $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 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 mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) 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 $30{\mu}g/ml$ of $HgCl_2$ 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).

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