A preliminary study on recombinant expression and function in vitro of proteasome activator REGγ / 军事医学科学院院刊

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.