Functional expression of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima in Escherichia coli. Authenticity and kinetic properties of the recombinant enzyme.
The gene coding for D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima has been cloned and functionally expressed in Escherichia coli. Some 90% of the coding gene was amplified by the polymerase chain reaction. The amplified gene segment was in full agreement with the previously determined amino acid sequence [Schultes, V., Deutzmann, R., Jaenicke, R. (1990) Eur. J. Biochem. 192, 25-31] and was completed by the insertion of synthetic linkers using site-directed mutagenesis. The resulting semisynthetic gene was expressed in high yield in the cytoplasm of E. coli and the recombinant enzyme was purified to homogeneity. It was shown to be identical with the enzyme isolated directly from T. maritima in all enzymatic and physicochemical properties investigated. The enzyme is allosterically inhibited by both D- and L-glyceraldehyde 3-phosphate at concentrations above 1 mM, and by arsenate at concentrations above 10 mM. The expressed protein restores the natural E. coli phenotype in a gap- strain, thus providing evidence that the hyperthermophilic protein can fold and associate to its native, functional state in its mesophilic host.
Tomschy
A
A
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Glockshuber
R
R
Jaenicke
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases