Xing Xiao-wei, Yuan Hong, Wang Wei
Cell Transplantation and Gene Therapy Center, the Third Xiangya Hospital of Central South University, Changsha 410013, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Aug;23(8):701-3.
To express SRG4, a novel mouse spermatogenesis gene in E.coli and purify its fusion protein.
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.
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.
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.
在大肠杆菌中表达新型小鼠精子发生基因SRG4并纯化其融合蛋白。
采用逆转录聚合酶链反应（RT-PCR）扩增位于SRG4 C末端的586 bp片段，该片段包含一个类似Sad1_UNC的结构域。将PCR产物克隆到pUCm-T载体中并测序。然后将cDNA片段亚克隆到带有6×组氨酸标签的原核表达载体PQE-30中。对PQE-30-SRG4进行测序并转化到大肠杆菌M15中。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导带组氨酸标签的融合蛋白表达。通过蛋白质免疫印迹法（Western blot）鉴定带组氨酸标签的融合蛋白，并用镍-亚氨基二乙酸琼脂糖（Ni-NTA Agarose）进行纯化。
成功构建重组质粒PQE-30-SRG4，并在大肠杆菌M15中表达。融合蛋白在IPTG诱导后4 - 5小时表达量达到最高。通过Western blot证实了带有6×组氨酸标签的融合蛋白SRG4，并经Ni-NTA Agarose纯化。
重组质粒PQE-30-SRG4可在大肠杆菌M15中表达。纯化的包含类似Sad1_UNC结构域的融合蛋白可用于研究SRG4在精子发生中的生物学功能。
