February 2019  Vol. 29  No. 2 J. Microbiol. Biotechnol. (2019), 29(2), 274–282 https://doi.org/10.4014/jmb.1704.04062 Research Article jmb Review Expression and Purification of Transmembrane Protein MerE from Mercury-Resistant Bacillus cereus Aatif Amin 1,3 * , Arslan Sarwar 3 , Mushtaq A. Saleem 3 , Zakia Latif 1 , and Stanley J. Opella 2 Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore-54590, Pakistan Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA Department of Microbiology, Faculty of Life Sciences, University of Central Punjab, Lahore-54000, Pakistan Introduction Mercury pollution is a global problem due to its toxic effects on humans and animals. Anthropogenic sources and activities, e.g., the release of untreated industrial wastewater from chlor-alkali plants, metal mining and the incineration of coal, are increasing mercury pollution day by day in the environment [1]. It is evident that the cytotoxic and neurotoxic effects to humans and animals are caused by inorganic (ionic) and organic forms of mercury [2]. In Pakistan, the maximum release of mercury (kg/year) is estimated as 1,625.11, 2,500, 21,120, 1,071.79, 5,779, and 4,637.55 through extraction processes, cement industries, chlor-alkali industries, intentional uses of mercury in consumer products, waste deposition/land filling and wastewater treatment plants, respectively, indicating 36,898.77 kg of total cumulative release of mercury per year [3]. Bacillus sp. RC607, identified for the first time as having chromosomal resistance against mercury, was isolated from a contaminated site, Boston Harbor, USA [4]. In later studies, strain RC607-like sequences were found in 74 Bacillus spp. isolated from Minimata Bay sediment, Japan, and also in several Russian environmental bacterial isolates. It was suggested that the possible global distribution of the RC607 mer operon had occurred, as Bacillus mer operon- bearing plasmids were found in several strains analyzed in the Russian study [5, 6]. Due to the presence of mercury in the environment and its toxic effects, microorganisms have evolved mercury Received: April 28, 2017 Revised: August 1, 2017 Accepted: August 4, 2017 First published online August 8, 2017 *Corresponding author Phone: +92-42-35952811; Fax: +92-42-99230481; E-mail: aatifamin93@gmail.com pISSN 1017-7825, eISSN 1738-8872 Copyright © 2019 by The Korean Society for Microbiology and Biotechnology Mercury-resistant (Hg ) 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 (HgCl ). 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 ) 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 HgCl 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). Keywords: 16S rRNA, Ni-NTA chromatography, pHLMerE, thrombin, Hg-detoxification