Overexpression of catalytically active yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase in Escherichia coli.
E. coli expression plasmids for yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase (EC 3.1.3.11) as wild-type enzyme and as lacZ fusion protein have been constructed from a pUC vector and a fragment of genomic yeast DNA. Both proteins were overexpressed in E. coli strain TG2 as enzymatically active soluble forms and purified to homogeneity. While the wild-type enzyme is indistinguishable from the authentic yeast enzyme with respect to molecular size, specific activity and kinetic properties, the lacZ fusion protein behaves differently. Being a tetramer like the wild-type enzyme, the specific activity of the purified fusion protein is lower than that of the native enzyme. In contrast to the wild-type enzyme the fusion fructose-1,6-bisphosphatase is not inhibited by excess substrate. Inhibition of the fusion protein by the most potent allosteric effectors of fructose-1,6-bisphosphatase, AMP and fructose 2,6-bisphosphate, is weaker than observed with the wild-type enzyme. The fusion protein but not the wild-type enzyme was found to bind to immobilized Procion Navy H-ER. This was employed to purify the fusion fructose-1,6-bisphosphatase by affinity chromatography. Polyclonal antibodies raised in rabbits against the fusion enzyme were found to cross-react with the wild-type enzyme, but not with E. coli proteins. Both fructose-1,6-bisphosphatases complement the fructose-1,6-bisphosphatase mutant DF656 of E. coli.
Institut für Biochemie, Bereich Medizin, Universität Leipzig, Germany.
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D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000096:biosynthesis* / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D006597:Fructose-Bisphosphatase; D005947:Glucose