Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera. (15th February 2021)

• Record Type: Journal Article
• Authors: Velappan, Nileena; Close, Devin; Hung, Li-Wei; Naranjo, Leslie; Hemez, Colin; DeVore, Natasha; McCullough, Donna K; Lillo, Antonietta M; Waldo, Geoffrey S; Bradbury, Andrew R M
• Is Part Of: Protein engineering, design & selection. Volume 34:(2021)
• Publication Date: 2021-02-15
• Subjects: crystal structure -- epitope binning -- improved expression and functionality -- intrinsically fluorescent scFv antibodies -- soluble scFv antibodies; Protein engineering -- Periodicals; 660.63
• DOI: 10.1093/protein/gzaa029
• Languages: English
• ISSNs: 1741-0126

Abstract

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.