Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

Journal Article · Sun Jan 01 04:00:00 EST 1995 · Applied and Environmental Microbiology

OSTI ID:75823

Diruggiero, J; Robb, F T ^[1]

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1. Univ. of Maryland Biotechnology Institute, Baltimore, MD (United States)

The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100{degrees}C, compared with 10.5 h for the enzyme purified from P. furiosus. 29 refs., 6 figs., 1 tab.

• DOE Contract Number: FG02-92ER20083
• OSTI ID: 75823
• Journal Information: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 1 Vol. 61; ISSN AEMIDF; ISSN 0099-2240
• Country of Publication: United States
• Language: English

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