Cloning, Expression and Purification of Yeast Aminotransferase

Abstract

Lysine is synthesized in Saccharomyces cerevisiae via the α‐aminoadipate pathway. α‐aminoadipate is made from α‐ketoadipate via the action of PLP‐dependent aminotransferase that has not been identified. Sequence alignment and a homology search for conserved amino acid residues were used to identify six aminotransferase genes in S. cerevisiae. On the basis of sequence homology the identified genes code for two aromatic aminotransferases, two aspartate aminotransferases, one kynurenine aminotransferase and one putative amiontransferase of unknown function. The cloned genes were obtained from the Harvard Institute of Proteomics Plasmid Repository. The genes were subcloned into expression vector pET16b. The expressed his‐tagged proteins have been purified using a Ni‐NTA affinity column. Kinetic characterization of these enzymes is at the early stages.