Kajino T, Ohto C, Muramatsu M, Obata S, Udaka S, Yamada Y, Takahashi H
Toyota Central Research & Development Laboratories, Inc., Nagakute, Aichi 480-1192, Japan.
Appl Environ Microbiol. 2000 Feb;66(2):638-42. doi: 10.1128/AEM.66.2.638-642.2000.
We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.
我们基于使用真菌蛋白二硫键异构酶（PDI）作为基因融合伴侣，开发了一种通用的短短芽孢杆菌表达和分泌系统。与PDI融合可提高异源蛋白的细胞外产量（免疫球蛋白G轻链提高8倍；香叶基香叶基焦磷酸合酶提高12倍）。与PDI连接可防止分泌蛋白聚集，从而使融合蛋白以可溶且具有生物活性的形式高水平积累。我们还表明，融合蛋白中PDI的二硫键异构酶活性负责抑制具有内二硫键的蛋白聚集，而即使将无内二硫键的蛋白与两个活性位点被破坏的突变型PDI融合，该蛋白的聚集也会被阻止，这表明PDI的另一种功能，如伴侣样活性，在PDI融合表达系统中协同防止了异源蛋白的聚集。
