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

Glutathione S-transferase (GST) from Schistosoma japonicum, which is w idely used for the production of fusion proteins in the cytoplasm of E scherichia coli, was employed as a functional fusion module that effec ts dimer formation of a recombinant protein and confers enzymatic repo rter activity at the same time. For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cyst atin, whose binding properties for papain were to be studied. The fusi on protein was secreted into the bacterial periplasm by means of the O mpA signal peptide to ensure formation of the two disulfide bonds in c ystatin. 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 expre ssion the soluble fusion protein could be isolated from the periplasmi c protein fraction. Purification to homogeneity was achieved in one st ep by means of an affinity column with glutathione agarose. Alternativ ely, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C-terminus. The GST moie ty of the fusion protein was enzymatically active and the kinetic para meters were determined using glutathione and 1-chloro-2,4-dinitrobenze ne 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 ef fect due to the dimer formation of GST. The quaternary structure was f urther confirmed by chemical crosslinking, which resulted in a specifi c 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 detect ion of binding interactions even in cases of low affinity.