| 王玉梅,赵心清,马昱澍,魏东芝.星海链霉菌卤化酶重组蛋白在大肠杆菌中可溶表达[J].,2014,54(3):278-284 |
| 星海链霉菌卤化酶重组蛋白在大肠杆菌中可溶表达 |
| Soluble expression of halogenase SinH from Streptomyces xinghaiensis in Escherichia coli |
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| DOI：10.7511/dllgxb201403003 |
| 中文关键词: 卤化酶 星海链霉菌 可溶表达 泛素标签 链霉菌分子伴侣 共表达体系 蛋白纯化 |
| 英文关键词: halogenase Streptomyces xinghaiensis soluble expression ubiquitin tag Streptomyces chaperonin coexpression system protein purification |
| 基金项目:华东理工大学生物反应器工程国家重点实验室开放课题资助项目；中国科学院热带海洋生物资源与生态重点实验室开放课题资助项目(LMB111002). |
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| 摘要点击次数: 2059 |
| 全文下载次数: 1989 |
| 中文摘要: |
| 大肠杆菌( Escherichia coli，E.coli )是表达异源蛋白的良好宿主，但常遇到蛋白表达产物不可溶的困难．在对来自星海链霉菌的卤化酶基因 sinH 进行大肠杆菌异源表达时，为克服蛋白表达产物可溶性低的问题，探讨了不同载体对蛋白可溶表达的影响．利用pET28a进行SinH表达时不能得到可溶的目标蛋白；使用含有泛素标签及内含肽的载体pHUIE实现了卤化酶蛋白SinH的可溶表达，但纯化后纯度不高；利用带有链霉菌分子伴侣蛋白基因的载体pET28a-SinH与pETcoco-pL1SL2在 E. coli BL21(DE3)中共表达，在30 ℃，0.1 mmol/L IPTG诱导条件下表达4 h，成功获得大量可溶蛋白，使用亲和层析(Ni-NTA)纯化，获得了较纯的目标蛋白SinH，上述研究结果为在大肠杆菌中进行其他链霉菌蛋白的可溶表达提供了参考． |
| 英文摘要: |
| Escherichia coli (E.coli) is a good host for expression of heterologous proteins but insoluble protein products are often obtained. In order to overcome the problem of low soluble expression halogenase gene sinH from Streptomyces xinghaiensis is expressed by E. coli the effect of different vectors on the solubility is discussed. No soluble target protein is detected when it is expressed using pET28a whereas by using expression vector pHUIE containing ubiquitin tag and intein soluble target protein is obtained and purified but the purity is low. The coexpression system containing pET28a-SinH and pETcoco-pL1SL2 which carries genes encoding Streptomyces chaperonin is constructed in E. coli BL21(DE3) and the optimized induction condition is established as 0.1 mmol/L IPTG for induction and expression time is 4 h under 30 ℃. The target protein is purified by affinity chromatography Ni-NTA and pure SinH protein is obtained. The above research results provide basis for further exploration of soluble protein expression of Streptomyces in E. coli . |
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