Expression, purification of proteasome subunit PSMB1 and application in screening of possible proteasome inhibitors.
- Author:
Cuiying FAN
1
;
Lixing FENG
;
Dongmei ZHANG
;
Suna PAN
;
Xuan LIU
;
De'an GUO
;
Jinling FAN
Author Information
1. College of Food & Engineering, Henan University of Science and Technology, Luoyang 471003, Henan, China.
- Publication Type:Journal Article
- MeSH:
Binding Sites;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Humans;
Proteasome Endopeptidase Complex;
biosynthesis;
genetics;
isolation & purification;
Proteasome Inhibitors;
isolation & purification;
Recombinant Proteins;
biosynthesis;
genetics;
metabolism;
Triterpenes;
metabolism;
Ubiquitin
- From:
Chinese Journal of Biotechnology
2012;28(2):233-242
- CountryChina
- Language:Chinese
-
Abstract:
Proteasome is a multi-subunit protease complex in eukaryocytes, and plays an important role in ubiquitin-proteosome pathway. Recombinant proteasome can be used to screen proteasome inhibitors. In this study, recombinant plasmid of pET28a-PSMB1 was constructed by inserting human proteasome catalytic subunit (PSMB1) cDNA (726 bp) into the prokaryotic expression vector pET28a(+), and transforming the plasmid into E. coli BL21(DE3) cells for expression. After overnight induction (1 mmol/L IPTG, 20 degrees C), an expected protein band with molecular weight of 27 kDa was observed on SDS-PAGE gel. The recombinant protein was then purified through affinity chromatography, and the purity is more than 95%. The amino acid sequence of the recombinant protein was validated by NanoLC-MS/MS. The data from in vitro BIAcore analysis showed that the recombinant PSMB1 could bind to celastrol. The binding affinity between PSMB1 and 10 micromol/L celastrol was more than 27RU.
