title: 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.
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.
authors.forename: Rongzhi
authors.initials: R
authors.lastname: Wang
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.
authors.forename: Shuangshuang
authors.initials: S
authors.lastname: Xiang
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.
authors.forename: Yonghui
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.
authors.forename: Qiuyu
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China wshyyl@sina.com.
authors.forename: Shihua
authors.initials: S
authors.lastname: Wang
mesh_terms: D000915:Antibody Affinity; D022801:Complementarity Determining Regions / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
chemical_list: D022801:Complementarity Determining Regions; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins