Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.

Autor:	Kowalsky CA; From the Department of Chemical Engineering and Materials Science., Faber MS; Department of Biochemistry and Molecular Biology., Nath A; Genetics Graduate Program., Dann HE; From the Department of Chemical Engineering and Materials Science., Kelly VW; From the Department of Chemical Engineering and Materials Science., Liu L; Department of Microbiology and Molecular Genetics, and., Shanker P; From the Department of Chemical Engineering and Materials Science., Wagner EK; the Department of Chemical Engineering, University of Texas, Austin, Texas 78712., Maynard JA; the Department of Chemical Engineering, University of Texas, Austin, Texas 78712., Chan C; From the Department of Chemical Engineering and Materials Science, Department of Biochemistry and Molecular Biology, Genetics Graduate Program., Whitehead TA; From the Department of Chemical Engineering and Materials Science, Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824 and taw@egr.msu.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2015 Oct 30; Vol. 290 (44), pp. 26457-70. Date of Electronic Publication: 2015 Aug 20.
DOI:	10.1074/jbc.M115.676635
Abstrakt:	Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu