The Application of a Novel Lytic System to the Recovery of Recombinant Proteins in E.coli.
The Application of a Novel Lytic System to the Recovery of Recombinant Proteins in E.coli.
复制标题
新型裂解系统在大肠杆菌中重组蛋白回收中的应用。
DOI：
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
Y. Gong
中科院分区：
文献类型：
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作者：
Yunpeng Yang;Q. Tong;T. Hu;Y. Qian;Sheng‐li Yang;Y. Gong
为了高效回收重组蛋白，利用T4溶菌酶通过切割细胞壁肽聚糖层中的特异性键来破坏细菌的特性，构建了温度敏感的裂解系统。通过构建低拷贝质粒 pSC-lys（pSC101 复制起点）并将其引入大肠杆菌来评估该系统。该质粒含有温度敏感的T4溶菌酶（LYS(ts)）基因，受三个串联tac启动子和LacI阻遏蛋白的控制，与其他携带pMB1、ColE1复制起点等的质粒兼容。在最佳裂解条件下，2--5倍浓缩培养物重悬于缓冲液A、β-半乳糖苷酶、重组分子伴侣GroEL和ZZ-融合鲑鱼六联降钙素中(Cal6) 在大肠杆菌中的释放简单、快速且定量，与 LYS(ts) 共表达。两种测试的重组蛋白保持了显着的产量。这种新型裂解系统将取代其他繁琐的裂解方法，可用于回收重组蛋白，以便在生物技术领域进一步纯化。
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, ColE1 replication origins, etc. Under the optimum lysis conditions, 2--5 fold condensed cultures resuspended in buffer A, beta-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.