title: In vitro molecular evolution of AL NEIBMs improved immunoglobulin (Ig) binding and antibody detection.
abstract: AL (SpA A domain-PpL B3 domain), LD5 (PpL B3 domain-SpA D domain-PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L-D-L) and LD3 (PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L) are novel evolved Ig binding molecules (NEIBMs) derived from the in vitro molecular evolution of combinatorial phage libraries displaying randomly rearranged Ig-binding domains of protein A and protein L. These molecules all showed novel Ig-binding properties of double-site binding to the VH3 and Vκ regions of human Ig Fab and high affinity for human IgM, which enhanced IgM detection in the anti-HCV ELISA assay. In this double-site binding, the A domain binds to the VH3 chain with low affinity. Whether the appropriate mutations in the A domain could improve this binding remains unknown. In this study, four combinatorial phage libraries displaying AL mutants with random mutations at different amino acid positions in the A domain were constructed. Seven AL mutant phages with significantly improved Ig binding activity were obtained from the phage library displaying AL mutants randomly mutated at positions 27 and 34 through human IgM-directed in vitro evolution. Two of the seven prokaryotically expressed AL mutants, AL (VV) and AL (KA), exhibited IgM and IgG binding activities equivalent to those of wild-type AL, whereas other mutants showed attenuated binding. However, after labeling with HRP, AL (VV) and AL (KA) showed improved IgM and IgG binding activity, which significantly improved the detection in the anti-HCV assay. Thus, the present study demonstrates that the binding properties of AL were successfully improved through phage-based molecular evolution, which could substantially contribute to the use of AL in antibody detection, and provides an example of successful protein engineering through in vitro molecular evolution.
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Ting
authors.initials: T
authors.lastname: He
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Ding
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Jiao-Jiao
authors.initials: JJ
authors.lastname: Feng
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Qiu-Li
authors.initials: QL
authors.lastname: Chen
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Huai-Min
authors.initials: HM
authors.lastname: Zhu
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Peng
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Rui
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
authors.forename: Xiang-Yu
authors.initials: XY
authors.lastname: Li
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. Electronic address: mingmeicao@163.com.
authors.forename: Ming-Mei
authors.initials: MM
authors.lastname: Cao
authors.affiliation: Department of Medical Microbiology and Parasitology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. Electronic address: pwpanwei@126.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Pan
mesh_terms: D001435:Bacteriophages / Q000235:genetics; D019020:Directed Molecular Evolution; D018937:Hepatitis C Antibodies / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D007075:Immunoglobulin M / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D015202:Protein Engineering; D013205:Staphylococcal Protein A / Q000235:genetics / Q000276:immunology
chemical_list: D018937:Hepatitis C Antibodies; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D013205:Staphylococcal Protein A
keywords: AL mutants; Antibody detection; Human IgM; NEIBM; Phage-based molecular evolution