Purification of a functional asparagine synthetase (PVAS2) from common bean (Phaseolus vulgaris), a protein predominantly found in root tissues

Asparagine synthetase (AS; EC 6.3.5.4) is encoded by two genes in the common bean (Phaseolus vulgaris L.). PVAS2, a cDNA coding for a type-II asparagine synthetase from this legume, was cloned into pUC18 and its protein product, a functional asparagine synthetase, was able to complement the Escherichia coli asparagine-auxotroph ER#4813. PVAS2 was subsequently cloned into the expression vector pGEXKG and overexpressed as a glutathione S-transferase (GST) fusion protein. A single-step affinity chromatography on glutathione-agarose was used to purify this recombinant AS from bacterial extracts, and rabbit polyclonal antibodies against PVAS2 were raised. Western blot analyses were performed to study the pattern of AS protein in different common bean organs under different growth conditions. Overall levels of AS protein in roots were independent of the light status, but they were higher in nitrate-fed plants. AS was also detected in the early stages of nodule development, before these become competent for nitrogen fixation, suggesting that AS is not involved in nitrogen translocation from the nodule, but in nodule growth processes. Accumulation of AS protein in the roots of mature plants, but not in the nodules, suggests that asparagine synthetase might play a functional role in nitrogen translocation from roots to the aerial organs in P vulgaris, a ureide legume. (C) 2004 Elsevier Ireland Ltd. All rights reserved.