On of a residue corresponding to Arg16CaiB by lysine (Lys13TBEA6) in V. paradoxus strain TBEA6 and an added glutamine residue (Gln196TBEA6) involving Leu195TBEA6 (corresponding to Leu184CaiB) and His197TBEA6 (corresponding to His185CaiB). Secondary structure analyses. The amino acid sequences of ActTBEA6 and its orthologues had been subjected to secondary structure prediction by the Jpred server (44) (see Fig. S2 inside the supplemental material). Resulting from their available solved crystal structures, formyl-CoA:oxalate CoA-transferase from E. coli (YfdW) (27, 28), its orthologue Frc from Oxalobacter formigenes (20, 26), and crotonobetainyl-CoA:carnitine CoA-transferase from E. coli (CaiB) (29, 30) as members of the CoA-transferase III family have been integrated for comparison. As shown in Fig. S2, the amino acid sequences of ActTBEA6, ActDPN7, and ActLB400 (YP_553419.1) are truncated by about 13 to 15 amino acid residues in comparison to all other incorporated sequences. Cloning on the putative acyl-CoA-transferase gene actTBEA6 into the vector pET22b( ), overexpression in E. coli Lemo21(DE3), and purification and characterization of your IKKε custom synthesis translational product. Based on nucleotide sequence information (GenBank accession no. ACC69030.2), native ActTBEA6 has a calculated molecular mass of 43.322 kDa (isotopically average), consists of 398 amino acids, and includes a calculated pI of 5.46. Within this study, the putative act gene of V. paradoxus strain TBEA6 was heterologously expressed as a His6-tagged protein employing the T7promoter/polymerase-based expression vector pET22b( ) and E. coli Lemo21(DE3) as the host strain. For this, the protein was equipped with an extra C-terminal His6 tag plus two vectorencoded amino acids (leucine and PAK review glutamate) and an N-terminal pelB signal sequence (22 amino acids plus 17 amino acids among pelB and the start off of act) for potential periplasmatic localization (see Supplies and Solutions) (see Fig. S1 within the supplemental material). Consequently, the heterologously expressed protein consisted of 445 amino acids, and it exhibited a theoretical molecular mass of 48.372 Da (isotopically typical) and a theoretical pI of 5.65. The overproduced enzyme was purified by immobilized metal chelate affinity chromatography to electrophoretic homogeneity (Fig. 4). Afterwards, ActTBEA6 was applied to analytical size exclusion chromatography. It revealed an apparent molecular mass of 96 3 kDa. This corresponds to a homodimer in the protein with a theoretical molecular mass of 96.7 kDa, such as the His6 tag as well as the further 39 amino acid residues on the Nterminal pelB signal sequence. The UV-visible spectrum (jb.asm.orgJournal of BacteriologySuccinyl-CoA:3-Sulfinopropionate CoA-TransferaseFIG four Purification of ActTBEA6 by affinity chromatography as revealed by SDS-PAGE. Lane 1, crude extract of cells; lane M, molecular mass marker; lane 2, soluble fraction right after centrifugation; lane 3, elution fraction soon after Ni-NTA affinity chromatography column; lane 4, pooled fractions recovered just after Superdex 200 HR size exclusion chromatography. Forty micrograms of protein was applied in lanes 1 and two. Lanes three and 4 were loaded with five g protein. The SDS gel was stained with Coomassie brilliant blue R.to 800 nm) of purified ActTBEA6 showed a single peak at 280 nm, which indicates the absence of any chromophoric cofactor. Act enzyme activity assays applying the heterologously expressed and purified protein. (i) Initial identification of an acceptable CoA-donor to get a.
