Cloning, Expression And Activity Of ChrT Gene From Serratia Sp. CQMUS2

Objective: To clone the full-length gene of chromate reductase T(ChrT) from Serratia sp. CQMUS2and construct the prokaryoticexpression vector and transform it into E.coli BL21(DE3) to expressprotein. After expression and purification, ChrT was analyzed the enzymeactivity and capacity of Cr(VI) reduction.Methods: The ChrT gene from Serratia sp. CQMUS2was isolated byPCR and high-efficiency thermal asymmetric interlaced PCR(hiTAIL-PCR), then was sequenced and analyzed the sequence features bybioinformatics software. The ChrT gene was recombined with vectorpMD19-T and pET-28a(+) and then transformed into E.coli BL21(DE3).The engineering bacteria was induced to express protein with IPTG, theexpression quantity was observed at different time; Periplasmic, solublesand inclusion were extracted and observed the form of expression andpositioning by SDS-PAGE electrophoresis; The recombinant protein ChrTwas purified by Ni-NTA affinity chromatography and the activity wasdetected through the change in absorbance at340nm of NADPH;Meanwhile, the ability of Cr(VI) reduction was estimated through thechange of Cr(VI) content in enzyme reaction. Results: The ChrT gene is an ORF of567bp that encodes a188-aaenzyme. Multiple sequence alignment and phylogenetic analysis showedthat76%are highly conservative and had the closest relationship withKlebsiella pneumoniae. Comparing ChrT to E. coli ChrR, the similarity of3D structure is up to85.6%. The engineered bacteria pET-28a (+)-ChrT/BL21induced by IPTG, the result indicated that the expression productsincreased gradually with time passed and achieved to the highest point afterinduced10h. The positioning for expression products and the solubleanalysis revealed that the recombinant protein was soluble and presented inthe cytoplasm. Purified by Ni-NTA, NADPH can be used by ChrT enzyme,so that the absorbance at340nm of NADPH decreased94.5%within5min.By the way, Cr(VI) can be reduced by ChrT, while Cr(VI) concentration ofthe experimental group is significantly lower than that of control group andthe emzyme activity was164U/mg.Conclusion: Successfully constructed engineered bacteria pET-28a(+)-ChrT/BL21have the ability to express the recombinant protein ChrTexactly, and the ChrT enzyme owned the ability on the reduction of Cr(VI),and this study provided engineered bacteria strains resources and geneticresources for further studying efficient Cr(VI)-removel engineeringbacteria.