Expression of Galactokinase As a Fusion Protein inEscherichia coliandSaccharomyces cerevisiae

Abstract

Plasmids are described that allow fusions between the Escherichia coli galK gene (coding for galactokinase) and any gene of interest. An example is given in which a galK gene, lacking the normal initiator methionine codon, is fused to various segments of the 5′ end of the tet^R gene of pBR322. The resulting plasmids complemented an E. coli galK mutant, and galactokinase activity was retained despite the addition of up to 250 foreign amino acids to the amino-terminus of the galactokinase polypeptide. In a second experiment, the galK gene was fused to the LEU2 gene of Saccharomyces cerevisiae. The resulting plasmid was able to complement a yeast GAL1⁻ mutant and galactokinase synthesis in yeast was controlled, via the LEU2 regulatory system, by the levels of leucine and threonine in the growth medium. The galK fusion plasmids should facilitate analysis of the control systems of a wide variety of genes in different organisms.