Expression, Purification And Activity Analysis Of Chicken USP1/UAF1Complex

Ubiquitin-proteasome pathway is one of the most important and highly efficientprotein degradation pathways. It is involved in many cell functions, such as theregulation of the cell cycle. The ubiquitin specific processing proteases （USPs） isknown to be the largest family which sharing the most diverse structure indeubiquitinating enzymes（DUBs） family and containing USP1, USP3, USP11andUSP28etc. USPs are able to remove ubiquitin or ubiquitin chains from target proteinsto eliminate or reverse the biological functions of these ubiquitinated proteins. UAF1（USP1-associated factor1） is an80kDa protein which is composed of N-terminaleight WD40repeats regions and C-terminal coiled-coil domain. UAF1forms acomplex with USP1（USP1/UAF1） and then increases USP1’s activity. TheUSP1/UAF1complex is a critical regulator of the DNA repair pathways bydeubiquitinating PCNA-ub and FANCD2-Ub （Fanconi Anemia D2） in human cells.To investigate the characterization and the role of UAF1/USP1complex in vivoand in vitro, the antibody specific for chicken UAF1protein is an essential tool.chUAF1was expressed using the pET28a prokaryotic expression system and purifiedfrom transformed E.coli cells. The polyclonal antibodies were generated byimmunizing Rex rabbits with purified chUAF1protein.Bac-to-Bac expression system was used to express soluble and activeUSP1/UAF1complex as well as each subunit. Their purities were more than85%andthe protein yields were0.2mg per gram cells.We measrued the activity and cleavage specificity of purified chUSP1GG680,681AA/chUAF1and chUSP1^GG680,681AA. Under our reaction condition, they wereable to cleave substrates Di-Ub WT（K63）by81%and77%, Di-Ub WT（K48）by99%and51%, Di-SUMO2WT by39%å'Œ4%, respectively.The results suggested chicken UAF1can forms the complex with chicken USP1 and stimulate USP1activity. chUSP1^GG680,681AA/chUAF1and chUSP1^GG680,681AAreferto cleave Di-Ub WT（K48）. They also can chop the Di-SUMO2WT chain, but theprecise underlying mechanisms remain to be determined.