Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

Abstract

Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.

Antibody Engineering for Pursuing a Healthier Future

“…Additionally, a method for rapid and effective high-affinity GFP-based antibody production corresponding to scFv was developed. It was demonstrated by inserting CDR3 into green fluorescence protein (GFP) loops for improved disease diagnosis and therapy (Wang et al, 2014b). Skp co-expressing scFv has high solubility and binding activity to antigen thermolabile hemolysin (TLH) (a pathogenic factor of Vibrio parahaemolyticus) and was developed by using pACYC-Duet-skp co-expression vector.…”

The structure of a GFP-based antibody (fluorobody) to TLH, a toxin fromVibrio parahaemolyticus

“…Alanine scanning showed that the Gln 1 , Arg and Pro residues of the CDR3 loop have the most prominent effects on antigen binding, and lysine scanning showed that the current Lys residue of the loop has the best binding affinity (Wang et al, 2014). In our structure, the Arg, Pro and Lys residues mainly help to stabilize the CDR loop structure, while residue Gln 1 is in a dynamic state and may be involved in direct interactions with the antigen.…”

“…For pathogen detection, a fluorobody was developed by inserting the L-CDR3 (light-chain complementary-determining region 3) of a high-affinity scFv into loop 9 of GFP (residues 171-176) (Wang et al, 2014). This fluorobody retains its fluorescence properties and binding specificity for thermolabile haemolysin (TLH), a toxin from Vibrio parahaemolyticus, a food-borne Gram-negative halophilic bacterium that causes lethal food-borne diseases and poses a serious threat to human and animal health all over the world (Burdette et al, 2008;Bresee et al, 2002).…”

A self-assembled protein nanoparticle serving as a one-shot vaccine carrier

“…For the Hepatitis B core antigen, the amino acid 144 served as an insertion site for heterologous antigen fusion (29). GFP protein has a thermal stable structure that constituted by 11 β-strands and 3 α-helices and some of the loops between strands have been explored as insertion sites for heterologous protein for various purposes (18,19,30). Among those candidates, loop173 linking strand 8 and strand 9 was chosen because it has a high capacity for foreign peptide insertion (Fig 3A) (30).…”

“…So it is conceivable that the protein sequences among fluorescent protein family members in the barrel shell are highly variable and fluorescent proteins possess desirable biophysical properties can be selected using directed evolution (11,(14)(15)(16)(17). The applications of fluorescent protein have been expanded into multiple areas beyond live imaging which includes serving as biological sensors (18,19), or detectors for protein-protein interaction or protein folding (20,21).…”