小麦蛋白质二硫键异构酶的结构域对其性质影响研究

Wheat protein disulfide isomerase (wPDI) contains four thioredoxin domains a-b-b'-a' and a C-terminal tail c. To study the effect of each domain of wPDI on its properties, eight truncated proteins of wPDI containing different domain combinations were constructed by subcloning. The target proteins were prepared after expression and purification, and then their enzymatic properties and protein electrophoresis were determined and analyzed. The results indicated that eight truncated proteins of wPDI were expressed in E.coli BL21(DE3). After metal chelate affinity chromatography and size exclusion chromatography, the deletants of wPDI were obtained with high purity. The results from activities assay suggested that all domains of wPDI contributed to its oxidoreductase activity and molecular chaperone activity, and tail c didn't affect its isomerase activity but retained the critical amino acid binding site for molecular chaperone activity. Moreover, the electrophoretic analysis of truanted proteins A and A' showed that the active-site cysteines in domain a were primarily in the oxidized state while those of domain a' were primarily in the reduced state. Therefore, these results provide the basis for deeplyunderstanding the function and property of wPDI.