Strep-tag II and Twin-Strep based cassettes for protein tagging by homologous recombination and characterization of endogenous macromolecular assemblies in Saccharomyces cerevisiae.

Abstract

Peptide sequences fused to a gene of interest facilitate the isolation of proteins or protein complexes from cell extracts. In the case of fluorescent protein tags, the tagged protein can be visually localized in living cells. To tag endogenous genes, PCR-based homologous recombination is a powerful approach used in the yeast Saccharomyces cerevisiae. This approach uses short, homologous DNA sequences that flank the tagging cassette to direct recombination. Here, we constructed a set of plasmids, whose sequences were optimized for codon usage in yeast, for Strep-tag II and Twin-Strep tagging in S. cerevisiae. Some plasmids also contain sequences encoding for a fluorescent protein followed by the purification tag. We demonstrate using the yeast pyruvate dehydrogenase (PDH) complex that these plasmids can be used to purify large protein complexes efficiently. We furthermore demonstrate that purification from the endogenous pool using the Strep-tag system results in functionally active complexes. Finally, using the fluorescent tags, we show that a kinase and a phosphatase involved in regulating the activity of the PDH complex localize in the cells' mitochondria. In conclusion, our cassettes can be used as tools for biochemical, functional, and structural analyses of endogenous multi-protein assemblies in yeast.