Construction of a yeast expression system with positive selection for gene insertion in the absence of a specific phenotype

A positive-selection system of cloned inserts in Escherichia coli has been devised using a streptomycin-resistant (Sm(r)) gene and streptomycin-dependent (Sm(d)) E. coli. A vector, pHA394, based on the yeast expression vector pAM82 has the Sm(r) gene, which inactivates streptomycin (Sm) and invalidates the streptomycin dependence, resulting in a very low transformation efficiency. Replacement of the Sm(r) gene by the recombinant vector allows high-frequency transformation. This system was applied to the lysozyme gene. After the yeast secretion signal was fused to the lysozyme gene using an intermediate vector, pHA474, the Sm(r) gene of pHA394 was replaced by the fusion gene, followed by transformation of Sm(d) E. coli. Analysis of the transformants showed that the plasmid gene contained 100% of the lysozyme gene.