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J Biotechnol. 2015 Oct 20;212:174-80. doi: 10.1016/j.jbiotec.2015.08.020. Epub 2015 Aug 28.

A versatile puromycin-linker using cnvK for high-throughput in vitro selection by cDNA display.

Author information

1
Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
2
School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai Nomi, Ishikawa 923-1292, Japan.
3
Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan. Electronic address: nemoto@fms.saitama-u.ac.jp.

Abstract

cDNA display is a powerful in vitro display technology used to explore functional peptides and proteins from a huge library by in vitro selection. In addition to expediting the in vitro selection cycle by using cDNA display, easy and rapid functional analysis of selected candidate clones is crucial for high-throughput screening of functional peptides and proteins. In this report, a versatile puromycin-linker employing an ultrafast photo-cross-linker, 3-cyanovinylcarbazole nucleoside, is introduced. Its utility for both in vitro selection using cDNA display and protein-protein interaction analysis using a surface plasmon resonance (SPR) system is described. Using this versatile puromycin-linker, we demonstrated the model in vitro selection of the FLAG epitope and a SPR-based assay to measure the dissociation constant between the B domain of protein A and immunoglobulin G. Improvement of the puromycin-linker as described herein should make the cDNA display method easier to utilize for design of protein or peptide based affinity reagents.

KEYWORDS:

3-Cyanovinylcarbazole nucleoside; Directed evolution; High-throughput system; In vitro selection; Puromycin; cDNA display

PMID:
26321074
DOI:
10.1016/j.jbiotec.2015.08.020
[Indexed for MEDLINE]
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