title: Engineered antibody CH2 domains binding to nucleolin: Isolation, characterization and improvement of aggregation.
abstract: Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a yeast display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this yeast display library by panning against nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val264and Leu309, in the β-sheet, in which replacement of both charged residues led to significant decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future.
authors.affiliation: College of Life Science, Xiamen University, Xiamen, Fujian, China.
authors.forename: Dezhi
authors.initials: D
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Gong
authors.affiliation: Faculty of Health Sciences, University of Macau, Taipa, Macau, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Department of General Surgery, The Fourth Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China. Electronic address: maronghai@163.com.
authors.forename: Xihai
authors.initials: X
authors.lastname: Chen
authors.affiliation: Protein Interaction Section, Cancer Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
authors.forename: Dimiter S
authors.initials: DS
authors.lastname: Dimitrov
authors.affiliation: Faculty of Health Sciences, University of Macau, Taipa, Macau, China. Electronic address: zhaoqi@alumni.cuhk.net.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D019295:Computational Biology / Q000379:methods; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066329:Protein Aggregates / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015003:Yeasts; D000097796:Nucleolin
chemical_list: D000941:Antigens; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D019151:Peptide Library; D010750:Phosphoproteins; D066329:Protein Aggregates; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097796:Nucleolin
keywords: Aggregation prone region; Antibody domain; CH2; Monoclonal antibody; Nucleolin; Yeast display