High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli. Production of polyclonal antibodies against the recombinant protein and expression studies in pine seedlings.
In a previous work we reported the molecular characterization of a glutamine synthetase (GS; EC 6.3.1.2.) complementary DNA from a woody plant (Cantón et al. (1993) Plant Mol. Biol. 22, 819-828). The isolated cDNA (pGSP114) encoding a Scots pine (Pinus sylvestris) cytosolic subunit, has been subcloned into the expression vector pET3c to overproduce the GS polypeptide in Escherichia coli cells. The recombinant GS protein showed the same molecular size as a native Scots pine GS subunit. Antibodies against the pET3c-GSP114 encoded protein were raised in rabbits by injecting purified preparations and specificity was determined by immunoprecipitation of GS activity present in pine crude extracts. In spite of the antibodies were able to recognize both cytosolic and chloroplastic GS in tomato plants, they were unable to immunodetect chloroplastic GS in green cotyledons of pine seedlings and cytosolic GS was the unique recognized polypeptide. Unlike to that found in other plant species, cytosolic GS was strongly expressed in green tissues as determined by protein and Northern analysis. Our results suggest a key role for cytosolic GS in photosynthetic tissues of conifers.
Canton
F R
FR
Laboratorio de Bioquímica y Biologia Molecular, Facultad de Ciencias, Universidad de Málaga, Spain.
Garcia-Gutierrez
A
A
Crespillo
R
R
Cánovas
F M
FM
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis* / Q000302:isolation & purification; D018551:Solanum lycopersicum / Q000201:enzymology; D041605:Pinus sylvestris; D014176:Protein Biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D000906:Antibodies; D018076:DNA, Complementary; D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase