Cloning, Expression And Characterization Of Succinyl-CoA Synthetase From M.tuberculosis

Succinyl-CoA synthetase (SCS) catalyzes the reversible reaction succinyl-CoA+NDP+P(i)——succinate+CoA+NTP (N denoting adenosine or guanosine). The enzyme consists of two different subunits, designatedαandβ. It is a key component of tricarboxylic acid cycle. Our previous comparative proteomics study revealed that Radix Ranuncoli Ternati, a clinical approved Traditional Chinese Medicine for tuberculosis treatment, down-regulated the SCS protein. The genes encoding SCSαand SCSβwere amplified from Mycobacterium tuberculosis genomic DNA, the PCR products were cloned into pET28a (+) and pET32a (+) vector, pSCSa-pET28 and pSCSβ-pET32 were constructed successfully. The recombinant plasmids were transformed into competent E. coli BL21 (DE3) separately or cotransformation. The recombinant fusion proteins were expressed in soluble form under IPTG. The supematant of sonicated recombinant BL21 (DE3) was collected and the fusion proteins were purified. Circular dichroism (CD) was used to determined the secondary structure feature of these proteins. The modeling of SCSa and SCSβof Mycobacterium tuberculosis was based on the tertiary structure of the two subunits of E.coli.SCS protein composd of SCSαand SCSβhas the enzymatic activity of SCS and catalyze the formation of succinyl-CoA. It also discribed the change of the succinic acid content of recombinant BL21 (DE3) during the expression of recombinant proteins. The results provide a basis for further validation its drug target value for TB drug discovery.