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

FEBS Letters

Crespillo, Remedios¹  Garcia-Gutierrez, Angel¹  Canton, Francisco R.¹  Cánovas, Francisco M.¹ 

[1] Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Málaga, E-29071 Málaga, Spain

关键词: Pinus;    Woody plants;    Glutamine biosynthesis;    Recombinant protein;    Protein expression;   

DOI  :  10.1016/0014-5793(96)00886-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown