摘要
目的构建P33ING1b核定位信号肽(NLS)与谷胱甘肽-S-转移酶(GST)重组的融合蛋白(GST-NLS)原核表达载体及建立GST-NLS纯化技术。方法先用RT-PCR从ING1基因中扩增编码NLS的cDNA片段,将其插入克隆质粒pbluescript-SK,再以克隆质粒NLS片段为模板,PCR扩增NLScDNA片段,并加入限制性酶切位点和终止子,进而将其插入原核表达质粒pGEX-5X-3,构建GST-NLS原核表达载体pGEX-5X-3-NLS,最后用IPTG诱导其在大肠杆菌(BL21)中表达,用谷胱甘肽-琼脂糖小珠亲和纯化表达的GST-NLS。结果酶切鉴定和测序分析显示,NLScDNA片段被成功插入pbluescript-SK多克隆位点;NLScDNA片段在pGEX-5X-3-NLS中的插入位点、碱基序列及读码框和终止子的次序完全正确,NLScDNA序列位于表达载体的GST序列下游,插入片段全长183bp。经异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-5-thiogalactoside,IPTG)诱导表达和亲和纯化,从负荷pGEX-5X-3-NLS质粒的BL21菌株中获得了31.57ku的GST-NLS。结论成功建立了重组GST-NLS的原核表达载体、表达菌株及诱导表达和纯化的方法学,为进一步研究p33ING1b入核运载机制提供了重要技术手段。
Objective To construct prokaryotic expression system for expressing GST-NLS which expressing a fusion protein of nuclear locating sequence （NLS） of P33^ING1 and glutathione-S-transferase （GST）, and establish purification technology of GST-NLS. Methods cDNA fragment encoding NLS ING1 was obtained by RT-PCR and was inserted into muhi-cloning site of pbluescfipt-SK. The NLS ING1 cDNA fragment was amplified from pbluescript-SK-NLS by PCR with suitable restriction enzyme sites and terminator being added at the ends of the amplified cDNA fragment. The amplified NLS cDNA fragment was inserted into multi-cloning site of pGEX-5X-3 to construct pGEX-5X-3-NLS. GST-NLS expression in E.coli BL21 was induced by isopropyl-β-3-D-5-thiogalactoside （IPTG）, and the GST-NLS ING1 fusion protein was purified with glutathione-sepharose. Results Restriction enzyme digesting and DNA sequencing confirmed that the cDNA sequence of NLS ING1 had been correctly inserted into the multi-cloning site of pbluescript-SK. Sequence analysis showed that in the prokaryotic expression cassette （pGEX-5X-3-NLS ING1）, NLS＇s inserting site, base sequence, reading frame and stop codon were completely correct. GST-NLS ING1 fusion protein （31.57 ku） was successful purified from E.coli BL21 after IPTG induction. Conclusion Prokaryotic expression vector for expressing GST-NLS ING1 was successfully constructed and the inducing condition and affinity purification method were optimized. It will provide an efficient and convenient tool for further investigation such as the mechanism of transporting P33^ING1b into nucleus.
出处
《国际生物医学工程杂志》
CAS
2006年第2期69-72,80,共5页
International Journal of Biomedical Engineering
基金
天津市高等学校科技发展基金资助项目(2004ZD06)
