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

TitleOptimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.
Publication TypeJournal Article
Year of Publication2015
AuthorsZhong, Nan, Loppnau Peter, Seitova Alma, Ravichandran Mani, Fenner Maria, Jain Harshika, Bhattacharya Anandi, Hutchinson Ashley, Paduch Marcin, Lu Vincent, Olszewski Michal, Kossiakoff Anthony A., Dowdell Evan, Koide Akiko, Koide Shohei, Huang Haiming, Nadeem Vincent, Sidhu Sachdev S., Greenblatt Jack F., Marcon Edyta, Arrowsmith Cheryl H., Edwards Aled M., and Gräslund Susanne
JournalPLoS One
Date Published2015
KeywordsAntibody Formation, Antigens, Cloning, Molecular, Humans, Immunoglobulin Fab Fragments, Peptide Library, Recombinant Proteins

<p>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.</p>

Alternate JournalPLoS ONE
PubMed ID26437229
PubMed Central IDPMC4593582
Grant ListGM072688 / GM / NIGMS NIH HHS / United States
GM094588 / GM / NIGMS NIH HHS / United States
HG006436 / HG / NHGRI NIH HHS / United States
MOP-136944 / / Canadian Institutes of Health Research / Canada

University of Toronto  UCSF  The University of Chicago  QB3  Chicago Biomedical Consortium