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ID 62307
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Yasui, Norihisa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan ORCID Kaken ID publons researchmap
Nakamura, Kazuaki Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
Yamashita, Atsuko Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan ORCID Kaken ID publons researchmap
Abstract
Synthetic binding proteins that have the ability to bind with molecules can be generated using various protein domains as non-antibody scaffolds. These designer proteins have been used widely in research studies, as their properties overcome the disadvantages of using antibodies. Here, we describe the first application of a phage display to generate synthetic binding proteins using a sweet protein, monellin, as a non-antibody scaffold. Single-chain monellin (scMonellin), in which two polypeptide chains of natural monellin are connected by a short linker, has two loops on one side of the molecule. We constructed phage display libraries of scMonellin, in which the amino acid sequence of the two loops is diversified. To validate the performance of these libraries, we sorted them against the folding mutant of the green fluorescent protein variant (GFPuv) and yeast small ubiquitin-related modifier. We successfully obtained scMonellin variants exhibiting moderate but significant affinities for these target proteins. Crystal structures of one of the GFPuv-binding variants in complex with GFPuv revealed that the two diversified loops were involved in target recognition. scMonellin, therefore, represents a promising non-antibody scaffold in the design and generation of synthetic binding proteins. We termed the scMonellin-derived synthetic binding proteins ‘SWEEPins’.
Keywords
combinatorial library
non-antibody scaffold
phage display
single-chain monellin
synthetic binding proteins
Note
This is an Accepted Manuscript of an article published by Oxford University Press.
Published Date
2021-1-2
Publication Title
The Journal of Biochemistry
Volume
volume169
Issue
issue5
Publisher
Oxford University Press (OUP)
Start Page
585
End Page
599
ISSN
0021-924X
Content Type
Journal Article
language
English
OAI-PMH Set
岡山大学
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Related Url
isVersionOf https://doi.org/10.1093/jb/mvaa147
Funder Name
Japan Society for the Promotion of Science
助成番号
15H05370
19H02841
17H03644