A High Through-put Platform for Recombinant Antibodies to Folded Proteins.
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.
From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158;
Michael
M
Hornsby
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Marcin
M
Paduch
¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada;
Shane
S
Miersch
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Annika
A
Sääf
From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158;
Tet
T
Matsuguchi
From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158;
Brian
B
Lee
From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158;
Karolina
K
Wypisniak
From the ‡Department of Pharmaceutical Chemistry University of California, San Francisco, California 94158;
Allison
A
Doak
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Daniel
D
King
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Svitlana
S
Usatyuk
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Kimberly
K
Perry
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Vince
V
Lu
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
William
W
Thomas
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Judy
J
Luke
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Jay
J
Goodman
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Robert J
RJ
Hoey
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Darson
D
Lai
¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada;
Carly
C
Griffin
¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada;
Zhijian
Z
Li
**Saskatchewan Cancer Agency, University of Saskatchewan, Saskatoon, S7N 4H4, Canada;
Franco J
FJ
Vizeacoumar
¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada;
Debbie
D
Dong
‖Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854;
Elliot
E
Campbell
‖Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854;
Stephen
S
Anderson
‡‡Structural Genomics Consortium, Toronto, M5G Il7, Canada.
Nan
N
Zhong
‡‡Structural Genomics Consortium, Toronto, M5G Il7, Canada.
Susanne
S
Gräslund
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637;
Shohei
S
Koide
¶Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, MG5 1L6, Canada;
Jason
J
Moffat
¶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.
Sachdev
S
Sidhu
§Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637; Jim.Wells@ucsf.edu koss@bsd.uchicago.edu sachdev.sidhu@utoronto.ca.
Anthony
A
Kossiakoff
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.
James
J
Wells
D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D057166:High-Throughput Screening Assays; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D014157:Transcription Factors