title: Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.
abstract: We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhong
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Loppnau
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Alma
authors.initials: A
authors.lastname: Seitova
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Mani
authors.initials: M
authors.lastname: Ravichandran
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Maria
authors.initials: M
authors.lastname: Fenner
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Harshika
authors.initials: H
authors.lastname: Jain
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Anandi
authors.initials: A
authors.lastname: Bhattacharya
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Ashley
authors.initials: A
authors.lastname: Hutchinson
authors.affiliation: Department of Biochemistry and Molecular Biology, Knapp Center for Biomedical Discovery, University of Chicago, 900 East 57th St., Chicago, IL 60637, United States of America.
authors.forename: Marcin
authors.initials: M
authors.lastname: Paduch
authors.affiliation: Department of Biochemistry and Molecular Biology, Knapp Center for Biomedical Discovery, University of Chicago, 900 East 57th St., Chicago, IL 60637, United States of America.
authors.forename: Vincent
authors.initials: V
authors.lastname: Lu
authors.affiliation: Department of Biochemistry and Molecular Biology, Knapp Center for Biomedical Discovery, University of Chicago, 900 East 57th St., Chicago, IL 60637, United States of America.
authors.forename: Michal
authors.initials: M
authors.lastname: Olszewski
authors.affiliation: Department of Biochemistry and Molecular Biology, Knapp Center for Biomedical Discovery, University of Chicago, 900 East 57th St., Chicago, IL 60637, United States of America.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
authors.affiliation: Department of Biochemistry and Molecular Biology, Knapp Center for Biomedical Discovery, University of Chicago, 900 East 57th St., Chicago, IL 60637, United States of America.
authors.forename: Evan
authors.initials: E
authors.lastname: Dowdell
authors.affiliation: Department of Biochemistry and Molecular Biology, Knapp Center for Biomedical Discovery, University of Chicago, 900 East 57th St., Chicago, IL 60637, United States of America.
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.affiliation: Department of Biochemistry and Molecular Biology, Knapp Center for Biomedical Discovery, University of Chicago, 900 East 57th St., Chicago, IL 60637, United States of America.
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
authors.affiliation: Terrence Donnelly Center for Cellular & Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors.forename: Haiming
authors.initials: H
authors.lastname: Huang
authors.affiliation: Terrence Donnelly Center for Cellular & Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Nadeem
authors.affiliation: Terrence Donnelly Center for Cellular & Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: Terrence Donnelly Center for Cellular & Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, MSB-4180, Toronto, ON M5S 1A8, Canada.
authors.forename: Jack F
authors.initials: JF
authors.lastname: Greenblatt
authors.affiliation: Terrence Donnelly Center for Cellular & Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
authors.forename: Edyta
authors.initials: E
authors.lastname: Marcon
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Aled M
authors.initials: AM
authors.lastname: Edwards
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.
authors.forename: Susanne
authors.initials: S
authors.lastname: Gräslund
mesh_terms: D000917:Antibody Formation / Q000502:physiology*; D000941:Antigens / Q000276:immunology*; D003001:Cloning, Molecular; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000276:immunology*
chemical_list: D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins