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Sci Rep. 2016 Mar 4;6:22575. doi: 10.1038/srep22575.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

Author information

1
School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
2
Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China.
3
Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67400, Illkirch, France.
4
New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
5
School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
6
School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
7
Institute for Complex Molecular Systems, Department of Biomedical Engineering Eindhoven University of Technology Den Dolech 2, 5600 MB Eindhoven, The Netherlands.
8
Department of Physics, Harvard University, Cambridge, MA02138, USA.

Abstract

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10(3)-10(6)). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

PMID:
26940078
PMCID:
PMC4778045
DOI:
10.1038/srep22575
[Indexed for MEDLINE]
Free PMC Article

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