Centers and Institutes
Published October 5, 2015 | Version v1
Journal article Open

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

Creators

  • 1. University of Toronto
  • 2. University of Chicago

Description

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.

Data availability

All relevant data are within the paper and its Supporting Information files, but additional information can be found at the Structural Genomics Consortium website: www.thesgc.org.

Files

journal.pone.0139695.pdf

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Additional details

Identifiers

DOI
10.1371/journal.pone.0139695
Other
oai:uchicago.tind.io:7575

Funding

AbbVie (United States)
Bayer Pharma AG
Boehringer Ingelheim
Canada Foundation for Innovation
Genome Canada
GlaxoSmithKline
Innovative Medicines Initiative
Janssen
Eli Lilly (Canada)
Merck & Co., Inc., Rahway, NJ, USA (United States)
Novartis Foundation
Ontario Ministry of Economic Development and Innovation
Pfizer
São Paulo Research Foundation
Takeda
Wellcome Trust
National Institute of General Medical Sciences
GM072688
National Institute of General Medical Sciences
GM094588
National Institutes of Health
Common Fund
Canadian Institutes of Health Research
MOP-136944

UChicago Information

Division(s)
Biological Sciences Division
Department(s)
Biochemistry and Molecular Biology
Center(s) or Institute(s)
Gwen Knapp Center for Lupus and Immunology Research