Kossiakoff Lab

University of Chicago

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

Research Area: Uncategorized Year: 2015
Type of Publication: Article
  • 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
Journal: Molecular & Cellular Proteomics Volume: 14
Number: 10 Pages: 2833-2847
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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