Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Hornsby M

PROVIDER: S-EPMC4597156 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Publications

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

Hornsby Michael M Paduch Marcin M Miersch Shane S Sääf Annika A Matsuguchi Tet T Lee Brian B Wypisniak Karolina K Doak Allison A King Daniel D Usatyuk Svitlana S Perry Kimberly K Lu Vince V Thomas William W Luke Judy J Goodman Jay J Hoey Robert J RJ Lai Darson D Griffin Carly C Li Zhijian Z Vizeacoumar Franco J FJ Dong Debbie D Campbell Elliot E Anderson Stephen S Zhong Nan N Gräslund Susanne S Koide Shohei S Moffat Jason J Sidhu Sachdev S Kossiakoff Anthony A Wells James J

Molecular & cellular proteomics : MCP 20150819 10

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 hig ...[more]

PMID: 26290498

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Project description:Obesity is one of the most common global health problems for all age groups with obese people at risk of a variety of associated health complications. Consequently, there is a need to develop new therapies that lower body fat without the side effects. However, obesity is a complex and systemic disease, so that in vitro results are not easily translatable to clinical situations. A promising way to circumnavigate these issues is to reposition already approved drugs for new treatments, enabling a more streamlined drug discovery process due to the availability of pre-existing pharmacological and toxicological datasets. Chemical libraries, such as the Prestwick Chemical Library of 1200 FDA approved drugs, are available for this purpose. We have developed a simple semi-automated whole-organism approach to screening the Prestwick Chemical Library for those compounds which reduce fat content using the model organism Caenorhabditis elegans. Our whole-organism approach to high-throughput screening identified 9 “lead” compounds that reduced fat within 2 weeks in the model. Further screening and analysis provided 4 “hit” compounds (Midodrine, Vinpocetine, Fenoprofen and Lamivudine) that showed significant promise as drugs to reduce fat levels. The effects of these candidates were found to further reduce fat content in nematodes where an nhr-49/PPAR mutation resulted in “overweight” worms. Upon unblinding the “hit” compounds, they were found to have recently been shown to have anti-obesity effects in mammalian models too. In developing a whole-animal chemical screen to identify pharmacological agents as potential anti-obesity compounds, we demonstrate how chemical libraries can be rapidly and relatively cheaply profiled for active hits. Using the nematode Caenorhabditis elegans thus enables drugs to be assessed for applicability in humans and provides a new incentive to explore drug repurposing as a feasible and efficient way to identify new anti-obesity compounds. C. elegans; Prestwick chemical library; Obesity; High-throughput screening, Repurposing drugs

Dataset Information

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

Publications

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

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