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Biotechnol J. 2017 Feb;12(2). doi: 10.1002/biot.201600454. Epub 2016 Dec 20.

A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping.

Author information

1
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
2
Protein Engineering and Antibody Technologies, Merck Serono, Merck KGaA, Darmstadt, Germany.

Abstract

Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full-length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full-length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark-derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A-GFP system. Yeast library screening of full-length vNAR variants which were detected via GFP expression yielded the same high-affinity binder that had previously been isolated by our group using the conventional epitope tag-based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost-friendly alternative to conventional epitope tag detections.

KEYWORDS:

2A peptide; IgNAR; Ribosomal skipping; Yeast surface display; vNAR

PMID:
27878965
DOI:
10.1002/biot.201600454
[Indexed for MEDLINE]

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