The coding sequence of the mature cyanogenic β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowski, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (London) 371, 578–586] 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 (Km) and the ratio between the maximal velocities and efficiency constants (Vmax., Vmax./Km) 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 [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 314, 142–152]. Molecular dissociation constants for the free enzyme (pKE1, pKE2) obtained with linamarin and PNP-Glc, and the enzyme substrate complexes (pKES1, pKES2) 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 (ki = 0.007 min-1) and apparent inhibition constants (Ki = 20 mM) comparable with those of the plant protein [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 315, 323–330]. In comparison with the native glycosylated plant protein, the recombinant protein was, however, found to be extremely sensitive to proteolysis and misfolding.
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Research Article|
February 15 1996
Co-purification from Escherichia coli of a plant β-glucosidase-glutathione S-transferase fusion protein and the bacterial chaperonin GroEL
Zsolt KERESZTESSY;
Zsolt KERESZTESSY
‡
*Department of Biochemistry and Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.
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Jane HUGHES;
Jane HUGHES
*Department of Biochemistry and Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.
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László KISS;
László KISS
†Institute of Biochemistry, Lajos Kossuth University, Egyetem tér 1, Debrecen, H-4010 Hungary
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Monica A. HUGHES
Monica A. HUGHES
§
*Department of Biochemistry and Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.
§To whom correspondence should be sent.
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Publisher: Portland Press Ltd
Received:
May 09 1995
Revision Received:
August 30 1995
Accepted:
October 18 1995
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 314 (1): 41–47.
Article history
Received:
May 09 1995
Revision Received:
August 30 1995
Accepted:
October 18 1995
Citation
Zsolt KERESZTESSY, Jane HUGHES, László KISS, Monica A. HUGHES; Co-purification from Escherichia coli of a plant β-glucosidase-glutathione S-transferase fusion protein and the bacterial chaperonin GroEL. Biochem J 15 February 1996; 314 (1): 41–47. doi: https://doi.org/10.1042/bj3140041
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