title: Overexpression, purification and biophysical characterisation of E. coli MerT.
abstract: Mercury resistance is the most widespread of all anti-microbial resistance occurring in a wide variety of Gram-negative and Gram-positive bacterial genera. The systems that are most studied and best understood are those encoded in mercury resistance (Mer) operons in Gram-negative bacteria. The mercury detoxification functions by the importation of highly toxic Hg(2+) into cytoplasm and enzymic reduction to volatile Hg(0). MerT is a small (13kDa) inner membrane protein involved in mercuric ion transport system. We have overexpressed recombinant 6His-tagged MerT from Escherichia coli in a native folded form and purified it to homogeneity in n-dodecyl-β-d-maltopyranoside (DDM) by immobilized metal affinity chromatography (IMAC). Circular dichroism showed that the protein is largely α-helical. Size-exclusion chromatography (SEC) in a variety of detergents showed that the protein exists in a multiple of oligomeric states as also confirmed by SEC coupled with multiple-angle light scattering.
authors.affiliation: University of Auckland, School of Biological Sciences, Private Bag 92019, Auckland, New Zealand.
authors.forename: Kien
authors.initials: K
authors.lastname: Ly
authors.affiliation: University of Auckland, School of Biological Sciences, Private Bag 92019, Auckland, New Zealand; Quantum Detectors Limited, Rutherford Appleton Laboratory, Harwell, Oxford, United Kingdom.
authors.forename: Liam
authors.initials: L
authors.lastname: O'Ryan
authors.affiliation: University of Auckland, School of Biological Sciences, Private Bag 92019, Auckland, New Zealand. Electronic address: a.mitra@auckland.ac.nz.
authors.forename: Alok K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D011994:Recombinant Proteins; C411362:dodecyl maltopyranoside; C094115:mercury transport protein, bacterial; D008320:Maltose
keywords: Circular dichroism; Membrane channel; Membrane protein overexpression; Mercury transport; SEC-MALLS