AUTHOR=Amin Aatif , Latif Zakia 

TITLE=Cloning, Expression, Isotope Labeling, and Purification of Transmembrane Protein MerF from Mercury Resistant Enterobacter sp. AZ-15 for NMR Studies

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.01250

DOI=10.3389/fmicb.2017.01250

ISSN=1664-302X

ABSTRACT=<p>Mercury resistant (Hg<sup>R</sup>) <italic>Enterobacter</italic> sp. AZ-15 was isolated from heavy metal polluted industrial wastewater samples near to districts Kasur and Sheikhupura, Pakistan. 16S rDNA ribotyping and phylogentic analysis showed 98% homology with already reported <italic>Enterobacter</italic> species. The <italic>mer</italic>F gene encoding transmembrane protein-MerF was amplified from genomic DNA and ligated into pET31b+ vector using restriction endonucleases, <italic>Sph</italic>I and <italic>Xho</italic>I. The genetic codons of <italic>mer</italic>F gene encoding cysteine residues were mutated into codons, translating into serine residues by site-directed mutagenesis. Ketosteroid isomerase (KSI), a fusion tag which is present in pET31b+ vector, was used in the expression of <italic>mer</italic>Fm gene. KSI was used to drive the target peptide (MerFm) into inclusion bodies so that the peptide yield and purity were increased. The stable plasmid pET31b+:<italic>mer</italic>Fm was transformed into C43(DE3) <italic>E.coli</italic> cells. The high expression of uniformly <sup>15</sup>N isotopically labeled-MerFm protein was induced with 1 mM IPTG. The purification of <sup>15</sup>N-MerFm recombinant protein by Ni-NTA and size exclusion chromatography involved an unfolding/refolding procedure. The two-dimensional HSQC NMR spectra of MerFm protein showed the purity and correct number of resonances for each amide. <sup>1</sup>H–<sup>15</sup>N HSQC NMR experiment also confirmed that no modification of the tryptophan residue occurred during cyanogen bromide cleavage. A small scale reservoir of Luria Bertani (LB) medium supplemented with 20 μg/ml of HgCl<sub>2</sub> showed 90% detoxification of Hg by <italic>Enterobacter</italic> sp. AZ-15. The accumulation of Hg on the cell surface of this strain was visualized by scanning electron microscopy (SEM) which confirmed its potential use in Hg-bioremediation.</p>