A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders
de Greef, Tom F. A.
Chong, ShaorongNote: Order does not necessarily reflect citation order of authors.
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CitationCui, N., H. Zhang, N. Schneider, Y. Tao, H. Asahara, Z. Sun, Y. Cai, et al. 2016. “A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.” Scientific Reports 6 (1): 22575. doi:10.1038/srep22575. http://dx.doi.org/10.1038/srep22575.
AbstractDrop-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 (103–106). 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.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:26318742
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