Glutathione S‐transferase can be used as a C‐terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli

Open Access
Publisher Website
Google Scholar
Abstract
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.