COPURIFICATION FROM ESCHERICHIA-COLI OF A PLANT BETA-GLUCOSIDASE-GLUTATHIONE S-TRANSFERASE FUSION PROTEIN AND THE BACTERIAL CHAPERONIN GROEL

The coding sequence of the mature cyanogenic beta-D-glucosidase (beta- D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot escul enta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowsk i, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (Lond on) 371, 578-586] was found to be tightly associated with the fusion p rotein and co-purified with it. In the presence of excess MgATP, relea se and folding of the fusion beta-glucosidase were demonstrated by a f ast increase in both linamarase and p-nitrophenyl-beta-D-glucopyranosi dase activity at a low protein concentration. A slow endogenous foldin g process was also detected by activity measurements. Michaelis consta nts (K-m) and the ratio between the maximal velocities and efficiency constants (V-max., V-max./K-m) for the hydrolysis of the natural subst rate, linamarin, and p-nitrophenyl beta-D-glucopyranoside (PNP-Glc) by the recombinant protein were found to be almost identical with those of the native glycosylated plant enzyme [Keresztessy, Kiss and Hughes (1994) Arch, Biochem. Biophys. 314, 142-152]. Molecular dissociation c onstants for the free enzyme (pK(1)(E), pK(2)(E)) obtained with linama rin and PNP-Glc, and the enzyme substrate complexes (pK(1)(ES), pK(2)( ES)) were also in accordance with that of the original protein. The re active substrate analogue N-bromoacetyl beta-D-glucosylamine inactivat ed the fusion enzyme according to pseudo-first-order kinetics with fir st-order rate constant (k(i) = 0.007 min(-1)) and apparent inhibition constants (K-i = 20 mM) comparable viith those of the plant protein [K eresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 315, 323-33 0]. In comparison with the native glycosylated plant protein, the reco mbinant protein was, however, found to be extremely sensitive to prote olysis and misfolding.