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.
Details
Title
Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins
Author
Zhong, Nan : University of Toronto Loppnau, Peter : University of Toronto Seitova, Alma : University of Toronto Ravichandran, Mani : University of Toronto Fenner, Maria : University of Toronto Jain, Harshika : University of Toronto Bhattacharya, Anandi : University of Toronto Hutchinson, Ashley : University of Toronto Paduch, Marcin : University of Chicago Lu, Vincent : University of Chicago Olszewski, Michal : University of Chicago Kossiakoff, Anthony A. : University of Chicago Dowdell, Evan : University of Chicago Koide, Akiko : University of Chicago Koide, Shohei : University of Chicago Huang, Haiming : University of Toronto Nadeem, Vincent : University of Toronto Sidhu, Sachdev S. : University of Toronto Greenblatt, Jack F. : University of Toronto Marcon, Edyta : University of Toronto Arrowsmith, Cheryl H. : University of Toronto Edwards, Aled M. : University of Toronto Gräslund, Susanne : University of Toronto
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.
Funding Information
AbbVie 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 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, HG006436 SGC Canadian Institutes of Health Research, MOP-136944
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