A PNA-mediated Whiplash PCR-based program for in vitro protein evolution

John A. Rose, Mitsunori Takano, Akira Suyama

研究成果: Article

3 引用 (Scopus)

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The directed evolution of proteins, using an in vitro domainal shuffling strategy was proposed in (J. Kolkman and W. Stemmer, Nat. Biotech. 19, 423 (2001). Due to backhybridization during parallel overlap assembly, however this method appears unlikely to be an efficient means of iteratively generating massive, combinatorial libraries of shuffled genes. Furthermore, recombination at the domainal level (30-300 residues) appears too coarse to effect the evolution of proteins with substantially new folds. In this work, the compact structural unit, or module (10-25 residues long), and the associated pseudo-module are adopted as the fundamental units of protein structure, so that a protein may be modelled as an N to C-terminal walk on a directed graph composed of pseudomodules. An in vitro method, employing PNA-mediated Whiplash PCR (PWPCR), RNA-protein fusion, and restriction-based recombination is then presented for evolving protein sets with high affinity for a given selection motif, subject to the constraint that each represents a walk on a predefined pseudo-module digraph. Simulations predict PWPCR to be a reasonably high efficiency method of producing massive, recombined gene libraries encoding for proteins shorter than about 600 residues.

元の言語English
ページ(範囲)47-60
ページ数14
ジャーナルLecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics)
2568
出版物ステータスPublished - 2003
外部発表Yes

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ASJC Scopus subject areas

  • Computer Science(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Theoretical Computer Science

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