| 赵志文 张峥 刘浩文 黄智刚 吴颖.不同标签辅助的IBTX原核表达纯化及活性鉴定[J].,2014,14(7):1207-1211 |
| 不同标签辅助的IBTX原核表达纯化及活性鉴定 |
| The Expression, Purification and Identification of IBTX inwith Different Tags |
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| DOI： |
| 中文关键词: 钾离子通道毒素 IBTX 标签蛋白 BK |
| 英文关键词: KTX IBTX Protein tags BK |
| 基金项目:国家重点基础发展规划项目（973 子项目）（2010CB529804） |
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| 摘要点击次数: 918 |
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| 中文摘要: |
| 目的：在原核体系中建立高效表达纯化IBTX(Iberiotoxin)的工艺，并比较不同标签对IBTX生物活性的影响。方法：利用引
物搭桥的方法，经PCR 扩增，获得IBTX 编码基因，以此为模板分别构建了表达质粒PET32a(+)-IBTX、pGex-6p-1-IBTX、
pDuet-MBP-IBTX，并将其转入E.coli(BL21)分别表达带有硫氧还原蛋白(TRX)、谷胱甘肽巯基转移酶(GST)、麦芽糖结合蛋白
(MBP)标签的IBTX 融合蛋白，在经过亲和层析、烟草蚀纹病毒(TEV)蛋白酶酶切、C18 反向层析纯化后真空冻干得到IBTX干粉，
以电生理实验检测其生物活性。结果：在三种标签帮助下通过原核体系表达出的IBTX 都能够特异性的阻断大电导Ca2+激活的
钾通道(BK)电流，其中MBP帮助折叠的m-IBTX 活性最佳。结论：建立了IBTX 在MBP 标签帮助下的原核表达纯化工艺，为进
一步研究蝎钾离子通道alha家族神经毒素及其突变体的原核表达奠定了基础。 |
| 英文摘要: |
| Objective:To establish the process of expressing and purifing the effective IBTX in E.coli, and compare the impact of
different protein tags on the bioactivity of the recombinant IBTX.Methods: The overlapping polymerase chain reaction (PCR) was used,
and the IBTX coding sequence was cloned into PET32a (+), pGex-6p-1 and pDuet-MBP vectors. The plasimid PET32a (+)-IBTX,
pGex-6p-1-IBTX and pDuet-MBP-IBTX were transformed into E.coli BL21 for the expression of IBTX fusion proteins. After the process
of affinity chromatography, protein cleavage by TEV and reversed phase chromatography by a semi-preparative C18 column, the IBTX
was lyophilized into powder and tested by the electrophysiological experiments.Results:All the IBTXs folded with the exsistence of the
three tags in E.coli could specially block the BK channel, and the IBTX folded by MBP (m-IBTX) showed the greatest acitvity.Conclusion:The process of IBTX expression and purification with the help of MBP in has been well established, which provides a
reference for the further research about getting the alpha-KTx family memebers and their variants in prokaryotic system. |
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