Co-purification from Escherichia coli of a plant β-glucosidase-glutathione S-transferase fusion protein and the bacterial chaperonin GroEL

Zsolt Keresztessy, Jane Hughes, László Kiss, Monica A. Hughes

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The coding sequence of the mature cyanogenic β-D-glucosidase (β-D-glucoside glucohydrolase, EC (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL was found to be tightly associated with the fusion protein and co-purified with it. In the presence of excess MgATP, release and folding of the fusion β-glucosidase were demonstrated by a fast increase in both linamarase and p-nitrophenyl-β-D-glucopyranosidase activity at a low protein concentration. A slow endogenous folding process was also detected by activity measurements. Michaelis constants (K(m)) and the ratio between the maximal velocities and efficiency constants (V(max.), V(max.)/K(m)) for the hydrolysis of the natural substrate, linamarin, and p-nitrophenyl β-D-glucopyranoside (PNP-Glc) by the recombinant protein were found to be almost identical with those of the native glycosylated plant enzyme. Molecular dissociation constants for the free enzyme (pK(E)1, pK(E)2) obtained with linamarin and PNP-Glc, and the enzyme substrate complexes (pK(ES)1, pK(ES)2) were also in accordance with that of the original protein. The reactive substrate analogue N-bromoacetyl β-D-glucosylamine inactivated the fusion enzyme according to pseudo-first-order kinetics with first-order rate constant (k(i) = 0.007 min-1) and apparent inhibition constants (K(i) = 20 mM) comparable with those of the plant protein. In comparison with the native glycosylated plant protein, the recombinant protein was, however, found to be extremely sensitive to proteolysis and misfolding.

Original languageEnglish
Pages (from-to)41-47
Number of pages7
JournalBiochemical Journal
Issue number1
Publication statusPublished - Feb 15 1996


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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