Expression and purification of recombinant NRL-Hsp90α and Cdc37-CRL proteins for in vitro Hsp90/Cdc37 inhibitors screening.
Hsp90 has emerged as a promising target for cancer treatment. Hsp90 interacts with co-chaperone Cdc37 to mediate the conformational maturation of its kinase client proteins. Screening small molecule inhibitors targeting Hsp90/Cdc37 might be a promising strategy for further cancer therapeutic. In order to establish a recombinant protein system, the novel cloning and purification of full-length human Hsp90α and Cdc37 from BL21 (DE3) Escherichia coli is described here. In this work, we cloned and expressed recombinant NRL-Hsp90α and Cdc37-CRL that represent the full-length human Hsp90α and Cdc37 fused with the split Renilla luciferase (RL) protein fragments. We also expressed the full-length RL protein as a control for inhibitors screening. Moreover, we confirmed that the interaction proteins were able to complement split luciferase fragments and show the RL activity when substrate was added. In comparison, two mutations NRL-Hsp90α (Q133A) and Cdc37 (R167A)-CRL retained only 20% of the complemented RL activities. Six small molecule compounds were tested using this recombinant system. Very interestingly, Sulforaphane, Withaferin A, Celastrol and EGCG all decreased the complemented NRL-Hsp90α/Cdc37-CRL activities in the concentration-dependent manner. In addition, neither Sulforaphane nor Withaferin A showed non-specific inhibition on full length RL activity. However, Celastrol and EGCG showed different RL inhibition levels. The other two compounds LBH-589 and 17-AAG showed neither NRL-Hsp90α/Cdc37-CRL nor RL inhibition activities. These results indicate that purified NRL-Hsp90α and Cdc37-CRL appeared as pure, stable and active conformation, and can be used as an in vitro bioluminescence system for Hsp90/Cdc37 inhibitors screening.
College of Life Science, Jilin University, 2699 Qianjin Street, Changchun 130021, China.
Jing
J
He
Xiaojia
X
Niu
Cheng
C
Hu
Hongyi
H
Zhang
Yingjie
Y
Guo
Yubin
Y
Ge
Guangyi
G
Wang
Yiqun
Y
Jiang
D000818:Animals; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D018833:Chaperonins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D060066:Protein Interaction Maps / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D048790:Renilla / Q000201:enzymology; D054852:Small Molecule Libraries / Q000494:pharmacology*
C498362:CDC37 protein, human; D018797:Cell Cycle Proteins; D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D049410:Luciferases, Renilla; D018833:Chaperonins
Cdc37-CRL; Expression; Inhibitors screening; NRL-Hsp90α; Purification