[Prokaryotic expression and purification of SPAG4L, a novel human testis gene].
OBJECTIVE
To express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.


METHODS
The 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.


RESULTS
The 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.


CONCLUSIONS
The recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.
Jiang
Xian-zhen
XZ
Department of Urology, Third Hospital of Central South University, Changsha 410013, China. kateljy@yahoo.com.cn
Yang
Ming-gang
MG
Xing
Xiao-wei
XW
D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C114799:SPAG4 protein, human