Expression of three plant glutamine synthetase cDNA in Escherichia coli

Abstract

Three cDNA clones encoding the closely related glutamine synthetase (GS) a, p and y polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant a, / 3 and y 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 M and y isoenzymes complemented a GS(g1nA)-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.

Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes

“…This seems a frequent situation for the heterologous expression of other GS genes in E. coli. In fact, a similar temperature-dependent production of recombinant GS was observed for the expression of P. vulgaris GS gene (Bennett and Cullimore 1990;Betti et al 2002), for two cytosolic GS isoforms from pine (De la Torre et al 2002) and for diVerent Arabidopsis cytosolic isoforms (Ishiyama et al 2004). Interestingly, other authors obtained active recombinant plant GS from E. coli cells cultured at 37°C (Sakakibara et al 1996;Choi et al 1999).…”

Targeting of glutamine synthetase to the mitochondria of transgenic tabacco

“…4B) it appears that the cleaved 7 isoenzymes in the mitochondria have a lower specific activity than the authentic 7 isoenzymes in the cytosol. This lower specific activity has also been shown for an N-terminally modified 7 polypeptide, expressed in E. coli [3].…”

“…It has a slightly different mobility on denaturing gels than GS polypeptides in tobacco leaves (M.P. Robbins, unpublished data) and has already been shown to assemble into an active, octameric GS isoenzyme when expressed in Escherichia coli [ 3 ].…”

High‐level expression of Pinus sylvestris glutamine synthetase in Escherichia coli

“…A strong immunoreactive signal was only present in solubilized insoluble fraction (results not shown) and therefore large-scale protein extraction for GS purification was carried out with the insoluble cell fraction. The expression of recombinant GS protein as insoluble aggregates has been previously reported by Bennet and Cullimore [1] who also showed that the degree of solubility of the expressed polypeptide was dependent on expression temperature.…”

“…A number of cDNA clones have been isolated from a variety of plant species (reviewed by Forde and Cullimore [10]; Marsolier and Hirel [22]) which have been very helpful in defining a detailed molecular analysis of GS in plants. The expression of plant GS cDNAs in E. coli allowed the functional rescue of glnA defective mutants [9,29,1]. The availability of such specific gene probes has also allowed the study of in vivo expression of individual genes.…”