Unit

UNIT 26.6 Site-saturation Mutagenesis: A Powerful Tool for Structure-Based Design of Combinatorial Mutation Libraries

  1. Evangelia G. Chronopoulou,
  2. Nikolaos E. Labrou

Published Online: 1 FEB 2011

DOI: 10.1002/0471140864.ps2606s63

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Chronopoulou, E. G. and Labrou, N. E. 2011. Site-saturation Mutagenesis: A Powerful Tool for Structure-Based Design of Combinatorial Mutation Libraries. Current Protocols in Protein Science. 63:26.6:26.6.1–26.6.10.

Author Information

  1. Agricultural University of Athens, Athens, Greece

Publication History

  1. Published Online: 1 FEB 2011
  2. Published Print: FEB 2011

Abstract

This unit describes a method for site-saturation mutagenesis (SSM) using PCR amplification with degenerate synthetic oligonucleotides as primers. SSM allows the substitution of predetermined protein sites against all twenty possible amino acids at once. Therefore, SSM is a powerful approach in protein engineering to characterize structure-function relationships, as well as to create improved protein variants. The procedure accepts double-stranded plasmid isolated from the dam+ E. coli strain. The procedure is simple, fast, efficient, and eliminates time-consuming subcloning and ligation steps. Curr. Protoc. Protein Sci. 63:26.6.1-26.6.10. © 2011 by John Wiley & Sons, Inc.

Keywords:

  • protein engineering;
  • mutagenesis;
  • combinatorial mutation libraries