Sp. NRC-1 merA was cloned into pET46 in frame using a sequence encoding an N-terminal His6 tag. The protein was wellexpressed in several E. coli strains (E. coli BL21(DE3), BL21 Codon Plus (DE3) RP, Tuner(DE3), and Arctic Express (DE3) RP) under many different circumstances, which includes concentrations of IPTG ranging from 10 M to 0.five mM, induction times ranging from three hours to overnight and temperatures ranging from ten to 37 . Nonetheless, the protein was insoluble in each case. That is a frequent phenomenon when proteins from halophiles are expressed in E. coli; halophilic proteins have evolved to be soluble and active beneath highsalt circumstances and do not necessarily fold effectively under the conditions on the E. coli cytoplasm.22, 23 We re-folded and re-constituted GCR from inclusion bodies working with a protocol that was thriving in re-folding a dihydrolipoamide reductase from Haloferax volcanii that had been expressed in E. coli.16 Inclusion bodies containing GCR had been dissolved in eight M urea and after that gradually diluted into a refolding buffer containing FAD and NAD at room temperature. GCR activity enhanced and after that leveled off inside 4 h. The re-constituted GCR was purified working with an 5-HT4 Receptor Purity & Documentation immobilized Cu2+ column (Figure 3A, Figure S2 (B) and Table S3 in the Supporting Information and facts). The His6-tagged GCR bound far more tightly to this column than the native enzyme (Figure S2 with the Supporting Information and facts), in all probability due to binding in the Nterminal His6 tag towards the resin. The purified protein lowered bis–glutamylcystine proficiently, using a kcat of 54 ?8 s-1, a KM of 1.1 ?0.1 mM, as well as a kcat/KM of four.9 (?0.9) ?104 M-1 s-1 (Figure 3B). These kinetic parameters agree properly with these reported by Sundquist and Fahey (kcat = 28 s-1, KM = 0.81 mM and kcat/KM = three.5 ?104 M-1s-1).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptBiochemistry. Author manuscript; accessible in PMC 2014 October 28.Kim and CopleyPagePurified GCR does not have mercuric reductase activity Considering that the gene encoding GCR is at present annotated as merA, we measured the mercuric reductase activity on the protein by following the oxidation of NADPH at 340 nm at space temperature.13 Assays had been carried out in 50 mM sodium phosphate, pH 6.7, containing 3 M KCl, 1.three M NaCl, 1 mM EDTA, 0.34 mM NADPH and as much as 1 mM HgCl2. No activity was observed over 5 min inside the presence of 0.six M enzyme, whereas GCR reductase activity was simply detectable over 30 s inside the presence of 0.06 M enzyme. Further, GCR activity was NTR1 Compound completely inhibited by addition of 1 mM HgCl2 (Figure S3 of the Supporting Data). This locating is constant with earlier reports showing that GCR is inhibited by quite a few divalent metal ions, like Cu2+, Co2+, and Hg2+.9 GCR belongs towards the pyridine nucleotide disulfide oxidoreductase loved ones The sequence of GCR has extremely substantial matches for the FAD/NAD(P) binding domain (PFAM, PF07992) and the dimerization domain (PFAM, PF02582) from the pyridine nucleotide-disulfide oxidoreductase household; E-values are 8.three ?10-19 and 3.43 ?10-13, respectively. PROSITE24 recognized a pattern for the class I pyridine nucleotide-disulfide oxidoreductase active internet site, and PRINTS25 reported a set of motifs as a grouped signature for the class I pyridine nucleotide disulfide reductases. Proteins inside the pyridine nucleotide-disulfide oxidoreductase loved ones catalyze reduction of a wide selection of disulfide substrates, and their sequences are very divergent (Figure four). On the other hand, all members from the household sha.