[Prokaryotic expression and purification of SRG4, a novel mouse spermatogenesis gene].
OBJECTIVE
To express SRG4, a novel mouse spermatogenesis gene in E.coli and purify its fusion protein.


METHODS
RT-PCR was used to amplify the 586 bp fragment that was located in SRG4 C-end and included a Sad1_UNC like domain. The PCR products were cloned into pUCm-T vectors and sequenced. Then the cDNA fragment was subcloned into PQE-30, a prokaryotic expression vector with 6xHis tag. PQE-30-SRG4 was sequenced and transformed into E.coli M15. The expression of histidine-tagged fusion protein was induced by IPTG. The histidine-tagged fusion protein was identified by Western blot and purified by Ni-NTA Agarose.


RESULTS
The recombinant plasmid PQE-30-SRG4 was constructed successfully and was expressed in E.coli M15. The expression of the fusion protein reached the top at 4-5 h after it was induced by IPTG. The fusion protein SRG4 with 6xHis tag was confirmed by Western blot and was purified by Ni-NTA Agarose.


CONCLUSIONS
The recombinant plasmid PQE-30-SRG4 can be expressed in E.coli M15. The purified fusion protein including a Sad1_UNC like domain can be used for studying the biological function of SRG4 in spermatogenesis.
Xing
Xiao-wei
XW
Cell Transplantation and Gene Therapy Center, the Third Xiangya Hospital of Central South University, Changsha 410013, China.
Yuan
Hong
H
Wang
Wei
W
D000818:Animals; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013091:Spermatogenesis / Q000235:genetics / Q000502:physiology*
D018076:DNA, Complementary; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C544269:SPATA4 protein, mouse