In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

Details

Serval ID
serval:BIB_8C3FE7AE3D35
Type
Article: article from journal or magazin.
Collection
Publications
Title
In vitro affinity screening of protein and peptide binders by megavalent bead surface display.
Journal
Protein Engineering, Design & Selection
Author(s)
Diamante L., Gatti-Lafranconi P., Schaerli Y., Hollfelder F.
ISSN
1741-0134 (Electronic)
ISSN-L
1741-0126
Publication state
Published
Issued date
2013
Volume
26
Number
10
Pages
713-724
Language
english
Abstract
The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

Keywords
Amino Acid Sequence, Cell Surface Display Techniques/methods, DNA/genetics, Genotype, Microspheres, Models, Molecular, Peptides/chemistry, Peptides/genetics, Peptides/metabolism, Phenotype, Polymerase Chain Reaction, Protein Biosynthesis, Protein Conformation, Proteins/chemistry, Proteins/genetics, Proteins/metabolism, Transcription, Genetic
Pubmed
Web of science
Open Access
Yes
Create date
02/02/2017 8:06
Last modification date
20/08/2019 14:50
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