Isolation, Characterization and Expression of a Cdna Clone Encoding Plastid Aspartate-Aminotransferase from Arabidopsis-Thaliana

A clone encoding aspartate aminotransferase (AAT, EC 2.6.1.1) was isolated from an Arabidopsis thaliana leaf cDNA library. This clone contains a 1365 bp open reading frame encoding a polypeptide of 49.8 kDa, designated Ataat1. The clone was shown to contain a chloroplastic isoenzyme as an in organellar protein import assay demonstrated that a radiolabelled transcription/translation product of 49.8 kDa was imported into viable pea chloroplasts and was subsequently processed to yield a mature protein of 45 kDa. The open reading frame corresponding to the predicted mature AAT was manipulated into an expression construct (pEC14). Transformed Escherichia coli cells containing pEC14 expressed up to 16 times more AAT activity than vector only controls, thus demonstrating conclusively that the clone encoded AAT.