Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins

Nobuhide Doi, Hideaki Takashima, Aiko Wada, Yuko Oishi, Tetsuya Nagano, Hiroshi Yanagawa

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 °C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.

Original languageEnglish
Pages (from-to)231-239
Number of pages9
JournalJournal of Biotechnology
Volume131
Issue number3
DOIs
Publication statusPublished - 2007 Sep 15

Fingerprint

Nucleic acids
Nucleic Acids
Display devices
Genotype
Proteins
Phenotype
Recovery
Messenger RNA
DNA
Drug Interactions
Antibodies
Peptides
Screening
Drug Receptors
Peptide Library
Enzyme Inhibitors
Gene Library
Proteomics
Epitopes
Polypeptides

Keywords

  • Directed evolution
  • Drug receptor
  • Protein engineering
  • Proteomics
  • Puromycin

ASJC Scopus subject areas

  • Biotechnology

Cite this

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins. / Doi, Nobuhide; Takashima, Hideaki; Wada, Aiko; Oishi, Yuko; Nagano, Tetsuya; Yanagawa, Hiroshi.

In: Journal of Biotechnology, Vol. 131, No. 3, 15.09.2007, p. 231-239.

Research output: Contribution to journalArticle

Doi, Nobuhide ; Takashima, Hideaki ; Wada, Aiko ; Oishi, Yuko ; Nagano, Tetsuya ; Yanagawa, Hiroshi. / Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins. In: Journal of Biotechnology. 2007 ; Vol. 131, No. 3. pp. 231-239.
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