The phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase genes from the thermophilic archaeon Sulfolobus solfataricus overlap by 8-bp. Isolation, sequencing of the genes and expression in Escherichia coli.
The overlapping genes encoding phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) from the hyperthermophilic archaeon Sulfolobus solfataricus have been cloned and sequenced. PCR primers based on highly conserved regions of different PGK sequences were used to isolate an internal region of the pgk gene. This was then used to screen a genomic library to isolate the full length pgk gene. A 2.5-kb BglII fragment of S. solfataricus DNA contained both the pgk gene and the gap gene immediately downstream. Unexpectedly, the pgk and gap genes were found to overlap by 8 bp, with the initiation codon of the gap gene preceding the termination codon of the pgk gene. Evidence that the two genes are co-transcribed was obtained by Northern-blot analysis. The S. solfataricus PGK amino acid sequence shows 43% and 45% identity to the PGK sequences of the Archaea Methanobacterium bryantii and Methanothermus fervidus, respectively. High level expression of the S. solfataricus PGK and GraP-DH in Escherichia coli was achieved, with heat treatment at 80 degrees C proving an effective first step in the purification of these recombinant enzymes from extracts of the E. coli host. Purified recombinant S. solfataricus PGK and GraP-DH showed half lives of 39 min and 17 h, respectively, at 80 degrees C. Unlike bacterial GraP-DH enzymes, S. solfataricus GraP-DH was able to use both NAD+ and NADP+ as cofactors, but exhibited a marked preference for NADP+.
Department of Biochemistry and Molecular Biology, University College, London, England.
C E
CE
Jones
T M
TM
Fleming
D A
DA
Cowan
J A
JA
Littlechild
P W
PW
Piper
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
D004274:DNA, Recombinant; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase