A High Through-put Platform for Recombinant Antibodies to Folded Proteins

Michael Hornsby; Marcin Paduch; Shane Miersch; Annika Sääf; Tet Matsuguchi; Brian Lee; Karolina Wypisniak; Allison Doak; Daniel King; Svitlana Usatyuk; Kimberly Perry; Vince Lu; William Thomas; Judy Luke; Jay Goodman; Robert J Hoey; Darson Lai; Carly Griffin; Zhijian Li; Franco J Vizeacoumar; Debbie Dong; Elliot Campbell; Stephen Anderson; Nan Zhong; Susanne Gräslund; Shohei Koide; Jason Moffat; Sachdev Sidhu; Anthony Kossiakoff; James Wells

doi:10.1074/mcp.O115.052209

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A High Through-put Platform for Recombinant Antibodies to Folded Proteins

Journal article

Open access

Peer reviewed

A High Through-put Platform for Recombinant Antibodies to Folded Proteins

Michael Hornsby, Marcin Paduch, Shane Miersch, Annika Sääf, Tet Matsuguchi, Brian Lee, Karolina Wypisniak, Allison Doak, Daniel King, Svitlana Usatyuk, …

Molecular & cellular proteomics, Vol.14(10), pp.2833-2847

10/2015

DOI: https://doi.org/10.1074/mcp.O115.052209

PMCID: PMC4597156

PMID: 26290498

Abstract

RNA, Small Interfering - genetics

Immunoglobulin Fab Fragments - genetics

Recombinant Proteins - genetics

Transcription Factors - genetics

High-Throughput Screening Assays

Recombinant Proteins - immunology

Escherichia coli - genetics

Antibodies - immunology

Antibodies - genetics

Antigens - immunology

Immunoglobulin Fab Fragments - immunology

Transcription Factors - immunology

Antigens - genetics

Protein Folding

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

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Title: A High Through-put Platform for Recombinant Antibodies to Folded Proteins
Creators: Michael Hornsby - From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158
Marcin Paduch - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Shane Miersch - ¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada
Annika Sääf - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Tet Matsuguchi - From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158
Brian Lee - From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158
Karolina Wypisniak - From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158
Allison Doak - From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158
Daniel King - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Svitlana Usatyuk - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Kimberly Perry - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Vince Lu - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
William Thomas - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Judy Luke - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Jay Goodman - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Robert J Hoey - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Darson Lai - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Carly Griffin - ¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada
Zhijian Li - ¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada
Franco J Vizeacoumar - Saskatchewan Cancer Agency, University of Saskatchewan, Saskatoon, S7N 4H4, Canada
Debbie Dong - ¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada
Elliot Campbell - ‖Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854
Stephen Anderson - ‖Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854
Nan Zhong - ‡‡Structural Genomics Consortium, Toronto, M5G Il7, Canada
Susanne Gräslund - ‡‡Structural Genomics Consortium, Toronto, M5G Il7, Canada
Shohei Koide - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Jason Moffat - ¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada
Sachdev Sidhu - ¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada; Jim.Wells@ucsf.edu koss@bsd.uchicago.edu sachdev.sidhu@utoronto.ca
Anthony Kossiakoff - §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637; Jim.Wells@ucsf.edu koss@bsd.uchicago.edu sachdev.sidhu@utoronto.ca
James Wells - From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158; Jim.Wells@ucsf.edu koss@bsd.uchicago.edu sachdev.sidhu@utoronto.ca
Publication Details: Molecular & cellular proteomics, Vol.14(10), pp.2833-2847
Date published: 10/2015
Grant note: 1S10OD012037 / NIH HHS P41 CA196276 / NCI NIH HHS 1U54HG006436 / NHGRI NIH HHS S10 OD012037 / NIH HHS U54 HG006436 / NHGRI NIH HHS
Academic Unit: Molecular Biology and Biochemistry (SAS)
Language: English
Resource Type: Journal article
Identifiers: 991031654695504646