Nucleic Acids Research Advance Access originally published online on June 15, 2009
Nucleic Acids Research 2009 37(16):e108; doi:10.1093/nar/gkp514
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Nucleic Acids Research, 2009, Vol. 37, No. 16 e108
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA–protein fusions
1Rational Evolutionary Design of Advanced Biomolecules, Saitama Small Enterprise Promotion Corporation, Saitama Industrial Technology Center, 3-12-18 Kamiaoki, Kawaguchi, Saitama 333-0844, 2Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Shinjuku-ku, 169-8555, 3Innovation Center for Start-ups, National Institute of Advanced Industrial Science and Technology, 2-2-2, Marunouchi, Chiyoda-ku, Tokyo, 100-0005, 4Janusys Corporation, No. 508 Saitama Industrial Technology Center, 3-12-18 Kamiaoki, Kawaguchi, Saitama 333-0844, 5Japan Science and Technology Agency, CREST, 5, Sanbancho, Chiyoda-ku, Tokyo, 102-0075, 6GenCom Corporation, 11 Minami-oya, Machida, 194-8511, 7Molecular and Cell Biology, 8Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566 and 9Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan
*To whom correspondence should be addressed. Tel: +81 48 858 3531; Fax: +81 48 858 3531; Email: nemoto{at}fms.saitama-u.ac.jp
Received January 4, 2009. Revised April 27, 2009. Accepted May 29, 2009.
We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.