Prokaryotic expression and purification of SPAG4L, a novel human testis gene.
- Author:
Xian-zhen JIANG
1
;
Ming-gang YANG
;
Xiao-wei XING
Author Information
- Publication Type:Journal Article
- MeSH: Carrier Proteins; biosynthesis; genetics; isolation & purification; Escherichia coli; genetics; metabolism; Humans; Male; Plasmids; Recombinant Fusion Proteins; biosynthesis; genetics; isolation & purification
- From: Journal of Southern Medical University 2010;30(9):2047-2050
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.
METHODSThe fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.
RESULTSThe recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.
CONCLUSIONThe recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.
