Purification and refolding of human alpha5-subunit (PSMA5) of the 20S proteasome, expressed as inclusion bodies in Escherichia coli.
The 20S proteasome is the central enzyme of nonlysosomal protein degradation in both the cytosol and nucleus. It is composed of 28 protein subunits which are arranged into four staggered heptameric rings. The outer rings consist of alpha-subunits which are responsible for binding of proteasome activators, inhibitors, and regulators. To better characterize human alpha5-subunit (PSMA5) of the 20S proteasome, we have established a high-efficiency Escherichia coli expression system. The DNA-coding sequence for the human PSMA5, which was subcloned into the vector pET-22b (+), has been expressed as inclusion bodies in E. coli BL21 (DE3). To produce the native PSMA5, straightforward protocols have been developed for refolding the human PSMA5 in the presence of surfactants using dilution refolding and size-exclusion chromatography matrix refolding methods. After refolding, recovery yields of about 20% were obtained, respectively, with purity above 95%. The human PSMA5 was detected by dynamic light scattering in refolding process, and the molecular weight of the final refolded product was measured using gel filtration chromatography, which indicates that the human PSMA5 exists mainly as tetramer.
Han
Yu-Gang
YG
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Liu
He-Li
HL
Zheng
Hong-Jin
HJ
Li
Sheng-Guang
SG
Bi
Ru-Chang
RC
D001483:Base Sequence; D002850:Chromatography, Gel; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D046988:Proteasome Endopeptidase Complex