Kwon W S, Da Silva N A, Kellis J T
Department of Chemical and Biochemical Engineering, University of California, Irvine 92717, USA.
Protein Eng. 1996 Dec;9(12):1197-202. doi: 10.1093/protein/9.12.1197.
An advantage of exporting a recombinant protein to the periplasm of Escherichia coli is decreased proteolysis in the periplasm compared with that in the cytoplasm. However, protein degradation in the periplasm also occurs. It has been widely accepted that the thermodynamic stability of a protein is an important factor for protein degradation in the cytoplasm of E.coli. To investigate the effect of the thermodynamic stability of an exported protein on the extent of proteolysis in the periplasm, barnase (an extracellular ribonuclease from Bacillus amyloliquefaciens) fused to alkaline phosphatase leader peptide was used as a model protein. A set of singly or doubly mutated barnase variants were constructed for export to the E.coli periplasm. It was found that the half-life of the barnase variants in vivo increased with their thermodynamic stability in vitro. A dominant factor for the final yield of exported barnase was not exportability but the turnover rate of the barnase variant. The yield of a stabilized mutant was up to 50% higher than that of the wild type. This suggests that exporting a protein to the periplasm and using protein engineering to enhance the stability can be combined as a strategy to optimize the production of recombinant proteins.
将重组蛋白输出到大肠杆菌周质的一个优势是，与细胞质相比，周质中的蛋白水解作用减弱。然而，周质中的蛋白质降解也会发生。人们普遍认为，蛋白质的热力学稳定性是大肠杆菌细胞质中蛋白质降解的一个重要因素。为了研究输出蛋白的热力学稳定性对周质中蛋白水解程度的影响，将与碱性磷酸酶前导肽融合的芽孢杆菌核糖核酸酶（一种来自解淀粉芽孢杆菌的细胞外核糖核酸酶）用作模型蛋白。构建了一组单突变或双突变的芽孢杆菌核糖核酸酶变体，用于输出到大肠杆菌周质中。研究发现，芽孢杆菌核糖核酸酶变体在体内的半衰期随着其体外热力学稳定性的增加而延长。输出的芽孢杆菌核糖核酸酶最终产量的一个主要因素不是输出能力，而是芽孢杆菌核糖核酸酶变体的周转率。稳定突变体的产量比野生型高出50%。这表明，将蛋白质输出到周质并利用蛋白质工程提高稳定性可以结合起来，作为优化重组蛋白生产的一种策略。
