Synthesis of Escherichia coli branched-chain amino acid aminotransferase in vitro in a coupled transcription-translation system

Synthesis of the Escherichia coli branched-chain amino acid aminotransferase (transaminase-B; EC 2.6.1.42), the product of the ilv E gene, has been accomplished in vitro in a coupled transcription-translation system directed by DNA from a plasmid known to contain the ilv EDA operon. The identification of the protein was determined by its comigration with purified transaminase B, both on two-dimensional gels and on slab gels containing exponential gradients of acrylamide. Also reported is a comparison between RNA-directed and DNA-directed (coupled) protein synthesis reactions for optimal magnesium concentration, pH, template concentration, and kinetics of incorporation.