Overproduction and control of the LEU2 gene product, beta-isopropylmalate dehydrogenase, in transformed yeast strains

Abstract

Two transformed yeast strains, 21D/pYT14-LEU2 and AH22/CV9-2, were found to produce beta-isopropylmalate dehydrogenase to such an extent that the enzyme constitutes 2 and 1%, respectively, of the total extractable protein. This is 30 and 15 times, respectively, above wild type level. beta-Isopropylmalate dehydrogenase was purified from strain 21D/pYT14-LEU2 to a purity of about 95% in essentially three steps. Strain 21D/pYT14-LEU2 carries the LEU2 gene on a vector that also contains the yeast 2-micrometers plasmid and therefore replicates autonomously, whereas strain AH22/CV9-2 carries multiple copies of the LEU2 gene integrated at its normal chromosomal location. Despite the different genetic arrangements, regulation of LEU2 gene expression by leucine and leucine plus threonine was normal. Immunotitration showed that the decrease in specific activity caused by leucine and threonine corresponded to a decrease in immunoreactive material.