[Expression, purification of proteasome subunit PSMB1 and application in screening of possible proteasome inhibitors].
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.
College of Food & Engineering, Henan University of Science and Technology, Luoyang 471003, Henan, China.
Cuiying
C
Fan
Lixing
L
Feng
Dongmei
D
Zhang
Suna
S
Pan
Xuan
X
Liu
De'an
D
Guo
Jinling
J
Fan
D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D053978:Pentacyclic Triterpenes; D046988:Proteasome Endopeptidase Complex / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D061988:Proteasome Inhibitors / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014315:Triterpenes / Q000378:metabolism; D025801:Ubiquitin
D053978:Pentacyclic Triterpenes; D061988:Proteasome Inhibitors; D011994:Recombinant Proteins; D014315:Triterpenes; D025801:Ubiquitin; C577981:PSMB1 protein, human; D046988:Proteasome Endopeptidase Complex; C050414:celastrol