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Updated: Jul 13, 2026
Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment
Published on: September 17, 2019
[Prokaryotic expression and purification of SRG4, a novel mouse spermatogenesis gene]
Xiao-wei Xing1, Hong Yuan, Wei Wang
1Cell Transplantation and Gene Therapy Center, the Third Xiangya Hospital of Central South University, Changsha 410013, China.
Aim:
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.
Conclusion:
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.
