title: Cloning, sequencing and expression of the gene encoding glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum.
abstract: The gene encoding glucose dehydrogenase has been identified by Southern analysis of doubly restricted genomic Thermoplasma acidophilum DNA, using two redundant 17-residue oligonucleotide probes reverse translated from protein N-terminal sequence data. A 1670-bp BamH1-EcoR1 restriction fragment was ligated into pUC19 and pUC18 (constructs pTaGDH1 and pTaGDH2, respectively) and cloned in Escherichia coli. The sequence of the whole fragment was determined, and a 1059-bp open reading frame identified as the gene encoding glucose dehydrogenase. Cell-free extracts from E. coli carrying construct pTaGDH1 displayed glucose dehydrogenase activity indistinguishable from controls, but extracts from cells carrying pTaGDH2 displayed a 600-fold increase in glucose dehydrogenase activity. For high-level expression and purification of native protein, the glucose dehydrogenase coding sequence was subcloned into pMEX8. Glucose dehydrogenase purified from E. coli expressing the pMEX8 construct was indistinguishable by SDS/PAGE, N-terminal amino-acid sequence and kinetic analysis from the native enzyme purified from Tp. acidophilum. The derived 352-amino-acid sequence shows less than 20% identity with the glucose dehydrogenases of Bacillus subtilis and Bacillus megaterium but, by comparison with other eubacterial and eukaryotic dehydrogenase sequences, a portion of its sequence has been tentatively identified as a cofactor-binding region.
authors.affiliation: Department of Biochemistry, University of Bath, England.
authors.forename: J R
authors.initials: JR
authors.lastname: Bright
authors.forename: D
authors.initials: D
authors.lastname: Byrom
authors.forename: M J
authors.initials: MJ
authors.lastname: Danson
authors.forename: D W
authors.initials: DW
authors.lastname: Hough
authors.forename: P
authors.initials: P
authors.lastname: Towner
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase