Simplifying the Detection of Surface Presentation Levels in Yeast Surface Display by Intracellular tGFP Expression

Steffen C Hinz; Adrian Elter; Julius Grzeschik; Jan Habermann; Bastian Becker; Harald Kolmar

doi:10.1007/978-1-4939-9853-1_12

Simplifying the Detection of Surface Presentation Levels in Yeast Surface Display by Intracellular tGFP Expression

Methods Mol Biol. 2020:2070:211-222. doi: 10.1007/978-1-4939-9853-1_12.

Authors

Steffen C Hinz¹, Adrian Elter¹, Julius Grzeschik¹, Jan Habermann¹, Bastian Becker¹, Harald Kolmar²

Affiliations

¹ Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
² Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany. kolmar@Biochemie-TUD.de.

PMID: 31625098
DOI: 10.1007/978-1-4939-9853-1_12

Abstract

Yeast surface display (YSD) is an ultra-high throughput method used in protein engineering. Protein-protein interactions as well as surface presentation on the yeast cell surface are verified through fluorophore-conjugated labeling agents.In this chapter we describe an improved setup for full-length surface presentation detection. To this end, we used a single open reading frame (ORF) encoding for the protein to be displayed and a 2A sequence and tGFP for an intracellular fluorescence signal. The 2A sequence allows the simultaneous generation of two separate proteins from the same ORF through ribosomal skipping. The entangled expression of the POI on the yeast surface and intracellular tGFP obviates the need for fluorescent staining steps.

Keywords: 2A peptide; GFP; Ribosomal skipping; T2A; Yeast surface display.

MeSH terms

Green Fluorescent Proteins* / biosynthesis
Green Fluorescent Proteins* / chemistry
Green Fluorescent Proteins* / genetics
Open Reading Frames*
Peptide Library*
Protein Engineering*
Saccharomyces cerevisiae* / chemistry
Saccharomyces cerevisiae* / genetics
Saccharomyces cerevisiae* / metabolism

Substances

Peptide Library
Green Fluorescent Proteins