BACKGROUND: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. RESULTS: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. CONCLUSION: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.

译文

背景:重组蛋白在宿主细胞中的过度产生通常会导致其错误折叠和聚集。通过共同过量生产单个分子伴侣来增加重组蛋白的溶解度的先前尝试仅部分成功。现在,我们评估了大肠杆菌细胞溶胶功能上协同的伴侣网络的联合过量生产对重组蛋白溶解性的影响。
结果:发现两步法显示出最强的溶解度增强。第一步,将四个分子伴侣系统GroEL / GroES,DnaK / DnaJ / GrpE,ClpB和小的HSP IbpA / IbpB与重组蛋白协同过量生产,以优化从头折叠。在第二步骤中,抑制蛋白质生物合成以允许伴侣蛋白介导的体内错误折叠和聚集的蛋白质的重新折叠。这种新颖的策略将64种不同异源蛋白质中70%的溶解度提高了42倍。
结论:这里提出的工程化大肠杆菌菌株和两步法导致各种重组蛋白的溶解度显着提高,应适用于生物技术中生产的多种靶蛋白。

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