PNA-mediated Whiplash PCR

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, su...

• Type: Conference
• Year: 2001
• Where: DNA
• Authors: John A. Rose, Russell J. Deaton, Masami Hagiya, Akira Suyama
• Keywords: Bioinformatics, DNA 2001, Domainal Shuffling Strategy, Parallel Overlap Assembly, Proteins