Abstract

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.

Document Type

Article

DOI

https://doi.org/10.1093/protein/gzaa029

Rights Information

© 2021 The Authors. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords

crystal structure, epitope binning, improved expression and functionality, intrinsically fluorescent scFv antibodies, soluble scFv antibodies

Publication Date

2-15-2021

Journal Title

Protein engineering, design & selection : PEDS

Share

COinS