Expression, purification of proteasome subunit PSMB1 and application in screening of possible proteasome inhibitors / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 233-242, 2012.
Article
in Chinese
| WPRIM
| ID: wpr-304497
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Triterpenes
/
Binding Sites
/
Recombinant Proteins
/
Ubiquitin
/
Proteasome Endopeptidase Complex
/
Escherichia coli
/
Proteasome Inhibitors
/
Genetic Vectors
/
Genetics
/
Metabolism
Type of study:
Diagnostic study
/
Screening study
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2012
Type:
Article
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