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. (C) 1998 Elsevier Science B.V.