A preliminary study on recombinant expression and function in vitro of proteasome activator REGγ
- VernacularTitle:蛋白酶体激活因子REGγ的重组表达及其体外功能的初步探讨
- Author:
Min, WU
;
Jing, NIE
;
Ling-qiang, ZHANG
;
Yuan, WANG
- Publication Type:Journal Article
- Keywords:
recombinant expression;
plasmids;
proteasome activator REGγ;
casein kinase-2 interacting protein-1;
polymase chain reaction
- From:Bulletin of The Academy of Military Medical Sciences
2010;34(1):5-7,11
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the expression of the fused proteasome activator REGγ(11S regulator complex gamma subunit) using gene recombination technology and to further study the interaction between REGγ and casein kinase-2 interacting protein-1(CKIP-1)in vitro.Methods Firstly, the full length cDNA fragment of REGγ was amplified through PCR using the plasmid pCMV-Myc-REGγ as template and subcloned into the prokaryotic expression vector pGEX-4T-2 before being transformed into E.coli BL21 cells. The protein expression was induced by isopropyl-β-D-thiogalactoside(IPTG) .Secondly, the protein expression was monitored by SDS-PAGE and Western blotting after ultrasonication. Finally, the GST Pull-down assay was performed to investigate the interaction between REGγ and CKIP-1 in vitro.Results The prokaryotic expression construct pGEX-4T-2-REGγ was generated successfully and confirmed by DNA sequencing. Expression analysis showed that the GST-REGγ protein was easily expressed and isolated mainly in the lysate supernatant after sonication and centrifugation. The GST Pull-down assay revealed the strong mutual interaction between REGγ and CKIP-1 in vitro.Conclusion The proteasome activator REGγ could interact with the negative regulator of osteoblastogenesis CKIP-1 in vitro and the current study has shed light on further investigations of their physiological relevance.
