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

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

doi:10.1016/j.crmeth.2022.100357

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

Cell Rep Methods. 2022 Dec 6;2(12):100357. doi: 10.1016/j.crmeth.2022.100357. eCollection 2022 Dec 19.

Authors

Ioannis Tsirkas¹, Tomer Zur¹, Daniel Dovrat¹, Amit Cohen¹, Lior Ravkaie¹, Amir Aharoni¹

Affiliation

¹ Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.

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.

Keywords: HA-tag; fluorescent imaging; scFv; yeast.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Coloring Agents / metabolism
Golgi Apparatus
Peroxisomes
Saccharomyces cerevisiae Proteins* / genetics
Saccharomyces cerevisiae* / genetics

Substances

Coloring Agents
Saccharomyces cerevisiae Proteins