Protein fluorescent labeling in live yeast cells using scFv-based probes

Ioannis Tsirkas, Tomer Zur, Daniel Dovrat, Amit Cohen, Lior Ravkaie, Amir Aharoni

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The fusion of fluorescent proteins (FPs) to endogenous proteins is a widespread approach for microscopic examination of protein function, expression, and localization in the cell. However, proteins that are sensitive to FP fusion or expressed at low levels are difficult to monitor using this approach. Here, we develop a single-chain fragment variable (scFv)-FP approach to efficiently label Saccharomyces cerevisiae proteins that are tagged with repeats of hemagglutinin (HA)-tag sequences. We demonstrate the successful labeling of DNA-binding proteins and proteins localized to different cellular organelles including the nuclear membrane, peroxisome, Golgi apparatus, and mitochondria. This approach can lead to a significant increase in fluorescence intensity of the labeled protein, allows C′-terminal labeling of difficult-to-tag proteins and increased detection sensitivity of DNA-damage foci. Overall, the development of a scFv-FP labeling approach in yeast provides a general and simple tool for the function and localization analysis of the yeast proteome.

Original languageEnglish
Article number100357
JournalCell Reports Methods
Volume2
Issue number12
DOIs
StatePublished - 19 Dec 2022

Keywords

  • HA-tag
  • fluorescent imaging
  • scFv
  • yeast

ASJC Scopus subject areas

  • Genetics
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Biochemistry
  • Radiology Nuclear Medicine and imaging
  • Biotechnology
  • Computer Science Applications

Fingerprint

Dive into the research topics of 'Protein fluorescent labeling in live yeast cells using scFv-based probes'. Together they form a unique fingerprint.

Cite this