Bennett M, Cullimore J
Department of Biological Sciences, University of Warwick, Coventry, England.
Eur J Biochem. 1990 Oct 24;193(2):319-24. doi: 10.1111/j.1432-1033.1990.tb19340.x.
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.
编码菜豆（法国豆）密切相关的谷氨酰胺合成酶（GS）α、β和γ多肽的三个cDNA克隆在大肠杆菌中进行了重组表达。GS表达质粒正确合成了重组α、β和γ多肽，这些多肽随后组装成具有催化活性的同八聚体同工酶。这些同工酶在离子交换和凝胶过滤色谱上的行为与其天然同源物相似。此外，α和γ同工酶补充了GS（glnA）缺陷型突变体，从而证明了它们在大肠杆菌中的生理活性。在GS多肽的定量表达及其补充大肠杆菌突变体的能力方面，观察到三种重组GS质粒之间存在差异。这些差异与多肽的溶解度程度相关，观察到溶解度取决于表达温度。在大肠杆菌中产生活性GS同工酶有助于分离和表征单个菜豆同八聚体GS同工酶。
