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

TitleA High Through-put Platform for Recombinant Antibodies to Folded Proteins.
Publication TypeJournal Article
Year of Publication2015
AuthorsHornsby, Michael, Paduch Marcin, Miersch Shane, Sääf Annika, Matsuguchi Tet, Lee Brian, Wypisniak Karolina, Doak Allison, King Daniel, Usatyuk Svitlana, Perry Kimberly, Lu Vince, Thomas William, Luke Judy, Goodman Jay, Hoey Robert J., Lai Darson, Griffin Carly, Li Zhijian, Vizeacoumar Franco J., Dong Debbie, Campbell Elliot, Anderson Stephen, Zhong Nan, Gräslund Susanne, Koide Shohei, Moffat Jason, Sidhu Sachdev, Kossiakoff Anthony, and Wells James
JournalMol Cell Proteomics
Date Published2015 Oct
KeywordsAntibodies, Antigens, Escherichia coli, High-Throughput Screening Assays, Immunoglobulin Fab Fragments, Protein Folding, Recombinant Proteins, RNA, Small Interfering, Transcription Factors

<p>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. </p>

Alternate JournalMol. Cell Proteomics
PubMed ID26290498
PubMed Central IDPMC4597156
Grant ListS10 OD012037 / OD / NIH HHS / United States
U54 HG006436 / HG / NHGRI NIH HHS / United States
1U54HG006436 / HG / NHGRI NIH HHS / United States
P41 CA196276 / CA / NCI NIH HHS / United States
1S10OD012037 / OD / NIH HHS / United States

University of Toronto  UCSF  The University of Chicago  QB3  Chicago Biomedical Consortium