Expression, purification and characterization of human ubiquitin-activating enzyme, UBE1.
UBE1 plays an important role in the first step of ubiquitin-proteasome pathway to activate ubiquitin. Both the structure and biochemical property research of human UBE1 protein, and the activity analysis of those enzymes which are related with ubiquitination pathway, are based on high purity of UBE1 protein. To obtain human UBE1 protein, the full length of human UBE1 was expressed in E. coli and purified by Ni-NTA superflow sepharose and strep-tactin sepharose which based on UB-UBE1 high-energy thioester bonded intermediate complex. It was demonstrated that purified UBE1 could activate and conjugate UB to ubiquitin-conjugating enzyme E2s. The purified large amount of UBE1 could be used for in vitro studies of ubiquitin pathway and structural studies.
Zheng
Mei
M
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, People's Republic of China.
Liu
Jing
J
Yang
Zhenxing
Z
Gu
Xing
X
Li
Fan
F
Lou
Tiantian
T
Ji
Chaoneng
C
Mao
Yumin
Y