Site‐saturation Mutagenesis: A Powerful Tool for Structure‐Based Design of Combinatorial Mutation Libraries

Evangelia G. Chronopoulou1, Nikolaos E. Labrou1

1 Agricultural University of Athens, Athens, Greece
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 26.6
DOI:  10.1002/0471140864.ps2606s63
Online Posting Date:  February, 2011
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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

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Table of Contents

  • Introduction
  • Basic Protocol 1: Site‐Saturation Mutagenesis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Site‐Saturation Mutagenesis

  • E. coli competent cells (e.g., Top‐10, XL‐1 Blue, DH5 alpha; Stratagene, Invitrogen, Promega)
  • LB medium (see recipe)
  • Mini‐prep solution I (see recipe)
  • Mini‐prep solution II (see recipe)
  • Ice
  • Mini‐prep solution III (see recipe)
  • Isopropanol
  • Ethanol
  • Sterile ddH 2O or TE buffer ( appendix 2A)
  • High‐fidelity Pfu DNA polymerase for PCR (e.g., Stratagene, Promega)
  • 10× Pfu DNA polymerase buffer
  • 10 mM dNTPs
  • DpnI restriction endonuclease (e.g., New England Biolabs)
  • LB plates with appropriate antibiotics (see recipe)
  • 15‐ml tubes (with caps)
  • 37°C shaking incubator
  • 2‐ml microcentrifuge tubes
  • Microcentrifuge
  • Paper towels
  • Vortex
  • PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for designing custom‐synthesized oligonucleotides (primers) for PCR ( appendix 4J), analyzing the PCR product on an agarose gel (Irwin and Janssen, ; Wilson, ; also see appendix 4F), and preparing miniprep plasmid DNA by alkaline lysis ( appendix 4C)
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Literature Cited

   Andreadeli, A., Platis, D., Tishkov, V., Popov, V., and Labrou, N.E. 2008. Structure‐guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+. FEBS J. 275:3859‐3869.
   Arkin, A.P. and Youvan, D.C. 1992. A combinatorial optimization procedure for protein engineering: Simulation of recursive ensemble mutagenesis. Proc. Natl. Acad. Sci. U.S.A. 89:7811‐7815.
   Dougherty, M.J. and Arnold, F.H. 2009. Directed evolution: New parts and optimized function. Curr. Opin. Biotechnol. 20:486‐491.
   Georgescu, R., Bandara, G., and Sun, L. 2003. Saturation mutagenesis. In Methods in Molecular Biology Vol. 231: Directed Evolution Library Creation (F.H. Arnold and G. Georgiou, eds.) pp. 75‐83. Humana Press, Totowa, N.J.
   Gerlt, A. and Babbitt, P.C. 2009 Enzyme (re)design: Lessons from natural evolution and computation. Curr. Opin. Chem. Biol. 13:10‐18.
   Goltermann, L., Larsen, M.S., Banerjee, R., Joerger, A.C., Ibba, M., and Bentin, T. 2010. Protein evolution via amino acid and codon elimination. PLoS One 26:e10104.
   Irwin, N. and Janssen, K.A. 2001. Gel Electrophoresis of DNA and Pulsed‐field Agarose Gel Electrophoresis. Molecular Cloning Vol. 1: A Laboratory Manual (J. Sambrook and D.W. Russell, eds.) pp. 5.1‐5.17. CSHL Press, New York.
   Jäckel, C., Kast, P., and Hilvert, D. 2008. Protein design by directed evolution. Annu. Rev. Biophys. 37:153‐173.
   Kegler‐Ebo, D.M., Docktor, C.M., and DiMaio, D. 1994. Codon cassette mutagenesis: A general method to insert or replace individual codons by using universal mutagenic cassettes. Nucleic Acids Res. 22:1593‐1599.
   Kotzia, G.A. and Labrou, N.E. 2009. Engineering thermal stability of L‐asparaginase by in vitro directed evolution. FEBS J. 276:1750‐1761.
   Kotzia, G.A.,Lappa, K., and Labrou, N.E. 2007. Tailoring structure‐function properties of L‐asparaginase: Engineering resistance to trypsin cleavage. Biochem. J. 404:337‐343.
   Labrou, N.E. 2010. Random mutagenesis methods for in vitro directed enzyme evolution. Curr. Protein Pept. Sci. 11:91‐100.
   Maynard, J.A., Chen, G., Georgiou, G., and Iverson, B.L. 2002. In vitro scanning–saturation mutagenesis. Methods Mol. Biol. 182:149‐163.
   Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. Curr. Protoc. Mol. Biol. 59:2.1.1‐2.1.10.
   Otten, L.G., Hollmann, F., and Arends, I.W. 2010. Enzyme engineering for enantioselectivity: From trial‐and‐error to rational design? Trends Biotechnol. 28:46‐54.
   Patrick, W.M. and Firth, A.E. 2005. Strategies and computational tools for improving randomized protein libraries. Biomol. Eng. 22:105‐112.
   Reetz, M.T. and Carballeira, J.D. 2007. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2:891‐903.
   Reetz, M.T., Kahakeaw, D., and Sanchis, J. 2009. Shedding light on the efficacy of laboratory evolution based on iterative saturation mutagenesis. Mol. Biosyst. 5:115‐122.
   Reidhaar‐Olson, J.F. and Sauer, R.T. 1988. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science 241:53‐58.
   Romero, P.A. and Arnold, F.H. 2009. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10:866‐876.
   Tseng, W.C., Lin J.W., Wei, T.Y., and Fang, T.Y. 2008. A novel megaprimed and ligase‐free, PCR‐based, site‐directed mutagenesis method. Anal. Biochem. 375:376‐378.
   Wang, J., Zhang, S., Tan, H., and Zhao, Z.K. 2007. PCR‐based strategy for construction of multi‐site‐saturation mutagenic expression library. J. Microbiol. Methods 71:225‐230.
   Wilson, K. 1997. Preparation of genomic DNA from bacteria. Curr. Protoc.Mol. Biol. 27:2.4.1‐2.4.5.
   Wilson, K. 2002. Preparation and analysis of DNA: Agarose gel electrophoresis. In Current Protocols in Molecular Biology Vol. 1: Short Protocols In Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.13‐2.14. John Wiley & Sons, Hoboken.
   Wong, T.S., Tee, K.L., Hauer, B., and Schwaneberg, U. 2004. Sequence saturation mutagenesis (SeSaM): A novel method for directed evolution. Nucleic Acids Res. 32:1‐8.
   Zheng, L., Baumann, U., and Reymond, J.L. 2004. An efficient one‐step sitedirected and site‐saturation mutagenesis protocol. Nucleic Acids Res. 32:1‐5.
Internet Resources
  When using the tool in your experiments, use the following citation: Ulrich Krauss and Thorsten Eggert (2005) Insilico.mutagenesis: a primer selection tool designed for sequence scanning applications used in directed evolution experiments. BioTechniques 39 (5): p679‐682.
  PrimerX: Automated design of mutagenic primers for site‐directed mutagenesis.
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