Journal
PLOS ONE
Volume 10, Issue 10, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0139695
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Funding
- AbbVie [1097737]
- Bayer Pharma AG
- Boehringer Ingelheim
- Canada Foundation for Innovation
- Genome Canada
- GlaxoSmithKline
- Innovative Medicines Initiative
- Janssen
- Lilly Canada
- Merck Co.
- Novartis Research Foundation
- Ontario Ministry of Economic Development and Innovation
- Pfizer
- Sao Paulo Research Foundation
- Takeda
- Wellcome Trust
- National Institute of General Medical Sciences of the National Institutes of Health [GM072688, GM094588]
- NIH Common Fund [HG006436]
- SGC
- Canadian Institutes of Health Research [MOP-136944]
- Thermo Fisher Scientific
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We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.
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