The Research On Mycobacterium Tuberculosis Prokaryotic Ubiquitin-like Protein (Pup)-Proteasome System's Protein Expression And Purification

Prokaryotic ubiquitin-like protein (Pup)-proteasome system, the essential mechanism for prokaryotic cellular protein degradation in Mycobacterium tuberculosis(Mtb).With the help of accessory factors, Dop(Deamidase of Pup), PafA(Proteasome accessory factor) and Mpa(Myco-bacterial proteasome ATPase), Pup covalently linked to the Lysε-NH2 in the target proteins and mediated the target protein degradation through the proteasome. The discovery of Pup-proteas-ome system revealed a novel mechanism of prokaryotic protein degradation, which is involved essential physiological function including the intermediary metabolism, information pathway, detoxification/virulence, cell wall and cell membrane formation and so on. Disruption of Pup-proteasome system can suppress the pathogenicity of Mtb. Therefore it is regarded as the new therapeutic target for tuberculosis. In the present paper, the progress in the study on mechanism and function of Pup-proteasome system is reviewed.In this study, the genes of pup, dop, pafA and fabD are cloned from Mtb with gst or his6 tag for purification. With recombinant plasmids pGEX-2T-his6-pup, pET21cc-fabD-his6, pET21cc-dop-his6, pET21 cc-pafA-his6 and pBAD-pafA-his6, proteins are expressed and purified from E.coli (BL21) by nature or denature protein purification methods. These proteins are identified by western blot. Preparation of polyclonal antibody against Pup and preliminary study on dop knockout in Mycobacterium smegmatis can give us more knowledge and insight into Pup-Proteasome system and prokaryotic celluar regulatory mechanism.