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Molecular Biotechnology

, Volume 56, Issue 11, pp 992–1003 | Cite as

Strep-tag II and Twin-Strep Based Cassettes for Protein Tagging by Homologous Recombination and Characterization of Endogenous Macromolecular Assemblies in Saccharomyces cerevisiae

  • Jay Rai
  • J. Kalyani Pemmasani
  • Andriy Voronovsky
  • Ida S. Jensen
  • Arulmani Manavalan
  • Jens R. Nyengaard
  • Monika M. Golas
  • Bjoern Sander
Research

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.

Keywords

Strep-tag II Fluorescent protein tags Pyruvate dehydrogenase complex Single-particle electron microscopy Kinase Phosphatase 

Notes

Acknowledgments

This work has been supported by the Danish Council for Independent Research, the Danish Center for Scientific Computing (DCSC), the Lundbeck Foundation, the Agnes og Poul Friis Foundation, and the Fru Astrid Thaysens Foundation to MMG and BS. ISJ is supported by a fellowship of the Graduate School of Health, Aarhus University. The Centre for Stochastic Geometry and Advanced Bioimaging is supported by the Villum Foundation.

Supplementary material

12033_2014_9778_MOESM1_ESM.pdf (590 kb)
Supplementary material 1 (PDF 590 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jay Rai
    • 1
    • 3
  • J. Kalyani Pemmasani
    • 1
  • Andriy Voronovsky
    • 2
  • Ida S. Jensen
    • 2
  • Arulmani Manavalan
    • 1
  • Jens R. Nyengaard
    • 1
    • 3
  • Monika M. Golas
    • 2
    • 3
  • Bjoern Sander
    • 1
    • 3
  1. 1.Stereology and EM Laboratory, Department of Clinical Medicine, Institute of Clinical MedicineAarhus UniversityAarhus CDenmark
  2. 2.Department of BiomedicineAarhus UniversityAarhus CDenmark
  3. 3.Centre for Stochastic Geometry and Advanced BioimagingAarhus UniversityAarhus CDenmark

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