N-ICE plasmids for generating N-terminal 3 × FLAG tagged genes that allow inducible, constitutive or endogenous expression in Saccharomyces cerevisiae.

Abstract

PCR-mediated homologous recombination is a powerful approach to introduce epitope tags into the chromosomal loci at the N-terminus or the C-terminus of targeted genes. Although strategies of C-terminal epitope tagging of target genes at their loci are simple and widely used in yeast, C-terminal epitope tagging is not practical for all proteins. For example, a C-terminal tag may affect protein function or a protein may get cleaved or processed, resulting in the loss of the epitope tag. Therefore, N-terminal epitope tagging may be necessary to resolve these problems. In some cases, an epitope tagging strategy is used to introduce a heterologous promoter with the epitope tag at the N-terminus of a gene of interest. The potential issue with this strategy is that the tagged gene is not expressed at the endogenous level. Another strategy after integration is to excise the selection marker, using the Cre-LoxP system, leaving the epitope tagged gene expressed from the endogenous promoter. However, N-terminal epitope tagging of essential genes using this strategy requires a diploid strain followed by tetrad dissection. Here we present 14 new plasmids for N-terminal tagging, which combines two previous strategies for epitope tagging in a haploid strain. These 'N-ICE' plasmids were constructed so that non-essential and essential genes can be N-terminally 3 × FLAG tagged and expressed from an inducible promoter (GAL1), constitutive promoters (CYC1 or PYK1) or the endogenous promoter. We have validated the N-ICE plasmid system by N-terminal tagging two non-essential genes (SET1 and SET2) and two essential genes (ERG11 and PKC1). Copyright © 2016 John Wiley & Sons, Ltd.