Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently

• Published in: Nature biotechnology Vol. 20; no. 12; pp. 1251 - 1255
• Main Authors: Ness, Jon E, Kim, Seran, Gottman, Andrea, Pak, Rob, Krebber, Anke, Borchert, Torben V, Govindarajan, Sridhar, Mundorff, Emily C, Minshull, Jeremy
• Format: Journal Article
• Language: English
• Published: New York, NY Nature 01.12.2002; Nature Publishing Group
• Subjects: Amino Acid Sequence; Amino acids; Amino Acids - chemistry; Amino Acids - genetics; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Biological and medical sciences; Biotechnology; Combinatorial Chemistry Techniques - methods; Design criteria; DNA Shuffling - methods; Enzymes; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Methods. Procedures. Technologies; Molecular Sequence Data; Mutation; Peptide Library; Protein engineering; Protein Engineering - methods; Proteins; Recombinant Proteins - genetics; Sequence Alignment - methods; Sequence Analysis, Protein - methods; Peptidases; Protein engineering; Recombination; Serine endopeptidases; Gene; Enzyme; Hydrolases; Subtilisin; Protein
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt754

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.