Simplifying the Detection of Surface Presentation Levels in Yeast Surface Display by Intracellular tGFP Expression
| Autor: | Adrian Elter, Steffen C. Hinz, Harald Kolmar, Jan Habermann, Bastian Becker, Julius Grzeschik |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Ribosomal skipping Chemistry Cell Protein engineering 01 natural sciences Fluorescence Yeast Green fluorescent protein Cell biology 03 medical and health sciences Open reading frame 030104 developmental biology medicine.anatomical_structure 010608 biotechnology medicine Intracellular |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493998524 |
| DOI: | 10.1007/978-1-4939-9853-1_12 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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