Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes, and complementation of a glnA mutant.
Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes.
Bennett
M
M
Department of Biological Sciences, University of Warwick, Coventry, England.
Cullimore
J
J
D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007887:Fabaceae / Q000201:enzymology*; D005816:Genetic Complementation Test; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010946:Plants, Medicinal; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D007527:Isoenzymes; D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase