This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Diruggiero, J. Articles by Robb, F. T. Search for Related Content PubMed PubMed Citation Articles by Diruggiero, J. Articles by Robb, F. T. Agricola Articles by Diruggiero, J. Articles by Robb, F. T.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol., 01 1995, 159-164, Vol 61, No. 1
Copyright © 1995, American Society for Microbiology

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

J Diruggiero and FT Robb
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202.

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.

This article has been cited by other articles:

• Cabello, P., Roldan, M. D., Moreno-Vivian, C. (2004). Nitrate reduction and the nitrogen cycle in archaea. Microbiology 150: 3527-3546 [Abstract] [Full Text]  
• Vetriani, C., Maeder, D. L., Tolliday, N., Yip, K. S.-P., Stillman, T. J., Britton, K. L., Rice, D. W., Klump, H. H., Robb, F. T. (1998). Protein thermostability above 100°C: A key role for ionic interactions. Proc. Natl. Acad. Sci. USA 95: 12300-12305 [Abstract] [Full Text]  
• Kujo, C., Ohshima, T. (1998). Enzymological Characteristics of the Hyperthermostable NAD-Dependent Glutamate Dehydrogenase from the Archaeon Pyrobaculum islandicum and Effects of Denaturants and Organic Solvents. Appl. Environ. Microbiol. 64: 2152-2157 [Abstract] [Full Text]  
• Krah, R., O'Dea, M. H., Gellert, M. (1997). Reverse Gyrase from Methanopyrus kandleri. RECONSTITUTION OF AN ACTIVE EXTREMOZYME FROM ITS TWO RECOMBINANT SUBUNITS. J. Biol. Chem. 272: 13986-13990 [Abstract] [Full Text]  
• Komori, K., Miyata, T., DiRuggiero, J., Holley-Shanks, R., Hayashi, I., Cann, I. K. O., Mayanagi, K., Shinagawa, H., Ishino, Y. (2000). Both RadA and RadB Are Involved in Homologous Recombination in Pyrococcus furiosus. J. Biol. Chem. 275: 33782-33790 [Abstract] [Full Text]