Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.
Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.
复制标题
基于伴侣的程序，以增加大肠杆菌中产生的可溶性重组蛋白的产量。
DOI：
10.1186/1472-6750-7-32
复制
发表时间：
2007-06-12
影响因子：
3.5
通讯作者：
中科院分区：
文献类型：
--
作者：
宿主细胞中重组蛋白的过量生产常常导致其错误折叠和聚集。先前通过共过量生产单个分子伴侣来提高重组蛋白溶解性的尝试仅取得了部分成功。我们现在评估了大肠杆菌细胞质中功能协同的分子伴侣网络的联合过量生产对重组蛋白溶解性的影响。
发现一个两步程序对溶解性的增强作用最强。第一步，四个分子伴侣系统GroEL/GroES、DnaK/DnaJ/GrpE、ClpB以及小分子热休克蛋白IbpA/IbpB与重组蛋白协同共过量生产，以优化新生折叠。第二步，抑制蛋白质生物合成，使分子伴侣能够在体内介导错误折叠和聚集的蛋白质重新折叠。这种新策略使所测试的64种不同异源蛋白中的70%的溶解性提高了多达42倍。
此处介绍的工程化大肠杆菌菌株和两步程序使多种重组蛋白的溶解性显著提高，并且应该适用于生物技术中生产的大量目标蛋白。
The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins. A two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize de novo folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold. The engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.