Browse > Article
http://dx.doi.org/10.4014/jmb.1704.04062

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)
Publication Information
Journal of Microbiology and Biotechnology / v.29, no.2, 2019 , pp. 274-282 More about this Journal
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).
Keywords
16S rRNA; Ni-NTA chromatography; pHLMerE; thrombin; Hg-detoxification;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Tamura K, Nei M, Kumar S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 101: 11030-11035.   DOI
2 Huang CC, Narita M, Yamagata T, Endo G. 1999. Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1. Gene 239: 361-366.   DOI
3 Hamlett N, Landale E, Davis B, Summers A. 1992. Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding. J. Bacteriol. 174: 6377-6385.   DOI
4 Barkay T, Miller SM, Summers AO. 2003. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol. Rev. 27: 355-384.   DOI
5 Kiyono M, Sone Y, Nakamura R, Pan-Hou H, Sakabe K. 2009. The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli. FEBS Lett. 583: 1127-1131.   DOI
6 Baneyx F, Mujacic M. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22: 1399.   DOI
7 Li M, Su Z-G, Janson J-C. 2004. In vitro protein refolding by chromatographic procedures. Protein Expr. Purif. 33: 1-10.   DOI
8 Tsumoto K, Ejima D, Kumagai I, Arakawa T. 2003. Practical considerations in refolding proteins from inclusion bodies. Protein Expr. Purif. 28: 1-8.   DOI
9 Yamaguchi H, Miyazaki M. 2014. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules 4: 235-251.   DOI
10 Bajorunaite E, Cirkovas A, Radzevicius K, Larsen KL, Sereikaite J, Bumelis V-A. 2009. Anti-aggregatory effect of cyclodextrins in the refolding process of recombinant growth hormones from Escherichia coli inclusion bodies. Int. J. Biol. Macromol. 44: 428-434.   DOI
11 Normand P. 1995. Utilisation des sequences 16S pour le positionnement phyletique d'un organisme inconnu. Oceanis 21: 31-56.
12 Amin A, Latif Z. 2017. Cloning, expression, isotope labeling, and purification of transmembrane protein MerF from mercury resistant Enterobacter sp. AZ-15 for NMR studies. Front. Microbiol. 8: 1250.   DOI
13 Amin A, Latif Z. 2017. Screening of mercury-resistant and indole-3-acetic acid producing bacterial-consortium for growth promotion of Cicer arietinum L. J. Basic Microbiol. 57: 204-217.   DOI
14 Park SH, Das BB, Casagrande F, Tian Y, Nothnagel HJ, Chu M, et al. 2012. Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491: 779.   DOI
15 Cook GA, Stefer S, Opella SJ. 2011. Expression and purification of the membrane protein p7 from hepatitis C virus. J. Pept. Sci. 96: 32-40.   DOI
16 Das BB, Park SH, Opella SJ. 2015. Membrane protein structure from rotational diffusion. Biochim. Biophys. Acta (BBA)-Biomembr. 1848: 229-245.   DOI
17 Elly CT. 1973. Dithizone procedure for mercury analysis. J. Water Pollut. Control Fed. 940-945.
18 Humaira K, Mohammad J, Mohammad I. 2005. A simple spectrophotometric determination of trace level mercury using 1,5-diphenylthiocarbazone solubilized in micelle. Anal. Sci. 21: 507-512.   DOI
19 Chang JS, Huang JC. 1998. Selective adsorption/recovery of Pb, Cu, and Cd with multiple fixed beds containing immobilized bacterial biomass. Biotechnol. Prog. 14: 735-741.   DOI
20 Sathyavathi S, Manjula A, Rajendhran J, Gunasekaran P. 2013. Biosynthesis and characterization of mercury sulphide nanoparticles produced by Bacillus cereus MRS-1. Indian J. Exp. Biol. 51: 973-978.
21 Santos-Gandelman JF, Cruz K, Crane S, Muricy G, Giambiagi-deMarval M, Barkay T, et al. 2014. Potential application in mercury bioremediation of a marine sponge-isolated Bacillus cereus strain Pj1. Curr. Microbiol. 69: 374-380.   DOI
22 Neely A, Garcia-Olivares J, Voswinkel S, Horstkott H, Hidalgo P. 2004. Folding of active calcium channel ${\beta}1b$-subunit by size-exclusion chromatography and its role on channel function. J. Biol. Chem. 279: 21689-21694.   DOI
23 Dash HR, Das S. 2015. Bioremediation of inorganic mercury through volatilization and biosorption by transgenic Bacillus cereus BW-03 (PW-05). Int. Biodeterior. Biodegrad. 103: 179-185.   DOI
24 Ma C, Marassi FM, Jones DH, Straus SK, Bour S, Strebel K, et al. 2002. Expression, purification, and activities of fulllength and truncated versions of the integral membrane protein Vpu from HIV-1. Protein Sci. 11: 546-557.   DOI
25 Clark EDB. 2001. Protein refolding for industrial processes. Curr. Opin. Biotechnol. 12: 202-207.   DOI
26 Han Y-G, Liu H-L, Zheng H-J, Li S-G, Bi R-C. 2004. Purification and refolding of human ${\alpha}5$-subunit (PSMA5) of the 20S proteasome, expressed as inclusion bodies in Escherichia coli. Protein Expr. Purif. 35: 360-365.   DOI
27 Steenhuisen F, Wilson SJ. 2015. Identifying and characterizing major emission point sources as a basis for geospatial distribution of mercury emissions inventories. Atmos. Environ. 112: 167-177.   DOI
28 Ouellette T, Destrau S, Ouellette T, Zhu J, Roach JM, Coffman JD, et al. 2003. Production and purification of refolded recombinant human IL-7 from inclusion bodies. Protein Expr. Purif. 30: 156-166.   DOI
29 Sone Y, Nakamura R, Pan-Hou H, Sato MH, Itoh T, Kiyono M. 2013. Increase methylmercury accumulation in Arabidopsis thaliana expressing bacterial broad-spectrum mercury transporter MerE. AMB Express. 3: 52.   DOI
30 Park SH, Opella SJ. 2010. Triton X-100 as the "short-chain lipid" improves the magnetic alignment and stability of membrane proteins in phosphatidylcholine bilayers for oriented-sample solid-state NMR spectroscopy. J. Am. Chem. Soc. 132: 12552-12553.   DOI
31 Nakamura K, Silver S. 1994. Molecular analysis of mercury-resistant Bacillus isolates from sediment of Minamata Bay, Japan. Appl. Environ. Microbiol. 60: 4596-4599.   DOI
32 Santos-Gandelman JF, Giambiagi-deMarval M, Muricy G, Barkay T, Laport MS. 2014. Mercury and methylmercury detoxification potential by sponge-associated bacteria. Antonie Leeuwenhoek 106: 585-590.   DOI
33 Abbas Z, Chaudary MN, Raza A, Mehmood A. 2012. Toxicity of mercury in different samples (water and soils) and its exposure in pakistan. Sci. Int. 24: 421-429.
34 Sedlmeier R, Altenbuchner J. 1992. Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans. Mol. Gen. Genet. 236: 76-85.   DOI
35 Bogdanova E, Bass I, Minakhin L, Petrova M, Mindlin S, Volodin A, et al. 1998. Horizontal spread of mer operons among Gram-positive bacteria in natural environments. Microbiology 144: 609-620.   DOI
36 Lund PA, Ford SJ, Brown NL. 1986. Transcriptional regulation of the mercury-resistance genes of transposon Tn501. J. Gen. Microbiol. 132: 465-480.
37 Moore MJ, Distefano MD, Zydowsky LD, Cummings RT, Walsh CT. 1990. Organomercurial lyase and mercuric ion reductase: nature's mercury detoxification catalysts. Acc. Chem. Res. 23: 301-308.   DOI
38 Oehmen A, Fradinho J, Serra S, Carvalho G, Capelo J, Velizarov S, et al. 2009. The effect of carbon source on the biological reduction of ionic mercury. J. Hazard. Mater. 165: 1040-1048.   DOI
39 Lu GJ, Son WS, Opella SJ. 2011. A general assignment method for oriented sample (OS) solid-state NMR of proteins based on the correlation of resonances through heteronuclear dipolar couplings in samples aligned parallel and perpendicular to the magnetic field. J. Magn. Reson. 209: 195-206.   DOI
40 Glendinning K, Macaskie L, Brown N. 2005. Mercury tolerance of thermophilic Bacillus sp. and Ureibacillus sp. Biotechnol. Lett. 27: 1657-1662.   DOI
41 Nakamura K, Hagimine M, Sakai M, Furukawa K. 1999. Removal of mercury from mercury-contaminated sediments using a combined method of chemical leaching and volatilization of mercury by bacteria. Biodegradation 10: 443-447.   DOI
42 Sinha A, Pant KK, Khare SK. 2012. Studies on mercury bioremediation by alginate immobilized mercury tolerant Bacillus cereus cells. Int. Biodeterior. Biodegradation 71: 1-8.   DOI