Sp. NRC-1 merA was cloned into pET46 in frame using a sequence encoding an N-terminal His6 tag. The protein was wellexpressed in various E. coli strains (E. coli BL21(DE3), BL21 Codon Plus (DE3) RP, Tuner(DE3), and Arctic Express (DE3) RP) below a number of circumstances, such as concentrations of IPTG ranging from 10 M to 0.five mM, ROR custom synthesis induction instances ranging from 3 hours to overnight and temperatures ranging from 10 to 37 . Nevertheless, the protein was insoluble in every case. This can be a popular phenomenon when proteins from halophiles are expressed in E. coli; halophilic proteins have evolved to be soluble and active below highsalt conditions and don’t necessarily fold properly below the situations in the E. coli cytoplasm.22, 23 We re-folded and re-constituted GCR from inclusion bodies making use of a protocol that was effective 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 then gradually diluted into a refolding buffer containing FAD and NAD at area temperature. GCR activity increased then leveled off within 4 h. The re-constituted GCR was purified employing an immobilized Cu2+ column (Figure 3A, Figure S2 (B) and Table S3 of your Supporting Information and facts). The His6-tagged GCR bound more tightly to this column than the native enzyme (Figure S2 on the Supporting Info), in all probability due to binding with the Nterminal His6 tag for the resin. The purified protein reduced bis–glutamylcystine efficiently, with a kcat of 54 ?eight s-1, a KM of 1.1 ?0.1 mM, and also a kcat/KM of four.9 (?0.9) ?104 M-1 s-1 (Figure 3B). These kinetic parameters agree effectively with these reported by Sundquist and Fahey (kcat = 28 s-1, KM = 0.81 mM and kcat/KM = 3.5 ?104 M-1s-1).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptBiochemistry. Author manuscript; obtainable in PMC 2014 October 28.Kim and CopleyPagePurified GCR does not have mercuric reductase activity Considering that the gene encoding GCR is presently 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 have been carried out in 50 mM sodium phosphate, pH six.7, containing 3 M KCl, 1.three M NaCl, 1 mM EDTA, 0.34 mM NADPH and up to 1 mM HgCl2. No activity was observed more than five min within the presence of 0.six M enzyme, whereas GCR reductase activity was quickly detectable more than 30 s inside the presence of 0.06 M enzyme. Further, GCR activity was fully inhibited by addition of 1 mM HgCl2 (Figure S3 with the Supporting Details). This discovering is constant with previous reports showing that GCR is inhibited by quite a few divalent metal ions, such as Cu2+, Co2+, and Hg2+.9 GCR belongs for the pyridine nucleotide disulfide oxidoreductase family members The sequence of GCR has extremely important matches for the FAD/NAD(P) binding domain (PFAM, PF07992) along with the dimerization domain (PFAM, PF02582) on the pyridine nucleotide-disulfide oxidoreductase household; E-values are eight.3 ?10-19 and three.43 ?10-13, respectively. PROSITE24 recognized a pattern for the class I pyridine nucleotide-disulfide oxidoreductase active website, and PRINTS25 reported a set of motifs as a grouped signature for the class I pyridine nucleotide disulfide reductases. Proteins in the pyridine nucleotide-disulfide oxidoreductase family members catalyze KLF MedChemExpress reduction of a wide selection of disulfide substrates, and their sequences are extremely divergent (Figure four). Nevertheless, all members from the family members sha.
