Tandem Affinity Purification Of Protein Interacted With Mycobacterium Smegmatis Ercc3.

Eukaryotic XPB/Ercc3 is a subunit of protein complex TFIIH which participate in transcription initiate, promotor clearance and nucleotide excision repair(NER) of DNA damage. XPB uses its ATP-dependent helicase activity and ssDNA-dependent ATPase activity to play a role in the opening of DNA double strand in transcription and remove of damage contained DNA fragment in NER. XPB also participate cell cycle regulation and other regulation pathway as a molecular sensor and responsor to a lot of cell pathways. In prokaryotic life Mycobacterium, we found the homologue of eukaryotic ercc3, but no other transcription and NER relative gene which interact and co-work with eukaryotic ercc3. Mycobacterium has its prokaryote transcription and NER system of itself. So the function of ercc3 gene in Mycobacterium is still to a question at issue.By a Mycobacterim recombineer system base on phage gene, we inserted TAP tag (protein A-TEV protease site-calmodulin binding protein) into C teminal of ercc3 gene in M.smegmatis genome. As a result, the recombinant M.smegmatis express ercc3-tap gene as the wild type ercc3 gene, since they are both simple copy and transcripted by the same promotor. Used the method of TAP(tandom affinity purification), we purified the Ercc3-TAP and its interaction protein from cell extraction. After SDS-PAGE and silver stain, the purification of interaction protein was identified by LC-MS.The LC-MS result revealed a list of possible interaction protein with Ercc3, include DNA-directed RNA polymeraseβ'andβsubunit (rpoC and rpoB), and recombinant protein recA. By in-vitro test such as farwestern and pull-down, we found Ercc3 interaction directly with RpoC in-vitro. And other experience found Ercc3 also interact with hypothetical protein ATPase and MSMEG₅705. But we didn't find distinct phenotype on ercc3-delected strain. From the data above, we can deduce that ercc3 (with its interaction partner) may participate in transcription initiation, particularly transcription in some special environment.