Tudyka T, Skerra A
Institut für Biochemie, Technische Hochschule, Darmstadt, Germany.
Protein Sci. 1997 Oct;6(10):2180-7. doi: 10.1002/pro.5560061012.
Glutathione S-transferase (GST) from Schistosoma japonicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time. For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cystatin, whose binding properties for papain were to be studied. The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin. The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST. Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction. Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose. Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C terminus. The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Furthermore, strong binding activity for papain was detected in an ELISA. The signal with the cystatin-GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST. The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size. Thus, engineered GST is suitable as a moderately sized, secretion-competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity.
日本血吸虫谷胱甘肽S-转移酶（GST）广泛用于在大肠杆菌细胞质中生产融合蛋白，被用作一种功能性融合模块，它能促使重组蛋白形成二聚体，并同时赋予酶报告活性。为此，GST通过一个柔性间隔区与硫醇蛋白酶抑制剂胱抑素的C末端相连，对胱抑素与木瓜蛋白酶的结合特性进行研究。融合蛋白借助OmpA信号肽分泌到细菌周质中，以确保胱抑素中两个二硫键的形成。通过替换GST中四个暴露的半胱氨酸残基中的三个，防止在氧化环境中形成错误的交联。利用四环素启动子严格控制基因表达，可从周质蛋白组分中分离出可溶性融合蛋白。通过使用谷胱甘肽琼脂糖亲和柱一步实现纯化至均一。或者，在融合蛋白的C末端附加Strep标签后，通过链霉亲和素亲和层析分离该蛋白。融合蛋白的GST部分具有酶活性，并以谷胱甘肽和1-氯-2,4-二硝基苯为底物测定动力学参数。此外，在酶联免疫吸附测定中检测到对木瓜蛋白酶有很强的结合活性。胱抑素-GST融合蛋白的信号比胱抑素本身高得多，这表明由于GST形成二聚体而产生了亲和力效应。通过化学交联进一步证实了四级结构，化学交联产生了一种分子大小为原来两倍的特异性反应产物。因此，工程化的GST适合作为一种中等大小、具有分泌能力的融合伙伴，它可以赋予目标蛋白双价性，即使在低亲和力情况下也能促进结合相互作用的检测。
