新型大肠杆菌裂解系统在重组蛋白回收中的应用(英文)  

The Application of a Novel Lytic System to the Recovery of Recombinant Proteins in E.coli

作　　者：杨运桂[1] 童芹[1] 胡泰山[1] 钱友存[1] 杨胜利[1] 龚毅[1] 

机构地区：[1]中国科学院上海生命科学研究院上海生物工程研究中心,上海200233

出　　处：《生物化学与生物物理学报》2000年第3期211-216,共6页

基　　金：国家高技术"863"计划资助项目!No .10 2 11 0 2 0 1&&

摘　　要：溶菌酶通过降解细菌细胞壁的肽聚糖裂解细菌 ,利用此特性 ,构建温度敏感的内源裂解系统 ,达到高效释放和回收重组蛋白的目的。构建了温度敏感的T4溶菌酶基因表达质粒 pSC lys(pSC10 1复制子 )和p15A lys(p15A复制子 ) ,质粒与工程菌构成了新型内源裂解系统 ,该系统可以与高拷贝复制子 (如 pMB1,ColE1)的重组蛋白表达质粒兼容。结果表明 ,裂解系统的最适裂解条件由三个要素构成 :pSC lys 工程菌、2～ 5倍浓缩培养物、裂解缓冲液 (2mmol/LEDTA ,5 0mmol/LTris HCl,pH 8.0 )。在最适裂解条件下 ,胞液 β 半乳糖苷酶 ,重组蛋白分子伴侣GroEL及ZZ融合鲑鱼降钙素六聚体均能简单、迅速、定量地释放到裂解液上清 ,两种重组蛋白的表达量均能维持原来的表达水平。此系统可以代替传统的菌体裂解方法 ,在重组蛋白纯化前的回收方面具有较大的应用价值。In order to efficiently recover recombinant proteins, a temperature sensitive lytic system was constructed on the basis of the feature that T4 lysozyme disrupts the bacteria through cutting specific bond in the peptidoglycan layer of cell wall. This system was evaluated by constructing and introducing a low copy plasmid pSC lys (pSC101 replication origin) into E.coli . The plasmid contained a temperature sensitive T4 lysozyme (LYS ts ) gene under the control of three tandem tac promoters and the LacI repressor, which is compatible with other plasmids carrying pMB1, Col E1 replication origins, etc. Under the optimum lysis conditions, 2—5 fold condensed cultures resuspended in buffer A, β galactosidase, recombinant chaperone GroEL and ZZ fusion salmon hexamic calcitonin (Cal6) in E.coli were released simply, rapidly, and quantitatively, as co expressed with LYS ts . The two tested recombinant proteins maintained their significant productions. Instead of other cumbersome lysising methods, this novel lytic system will be useful in recovery of recombinant proteins for further purification in the field of biotechnology.

关 键 词：大肠杆菌 T4溶菌酶 裂解系统 重组蛋白 回收 

分 类 号：R378.21[医药卫生—病原生物学]