Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras

Mary F. Farrow1, Frances H. Arnold1

1 Caltech, Pasadena, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 26.2
DOI:  10.1002/0471140864.ps2602s61
Online Posting Date:  August, 2010
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Abstract

Recombination of distantly related and nonrelated genes is difficult using traditional PCR‐based techniques, and truncation‐based methods result in a large proportion of nonviable sequences due to frame shifts, deletions, and insertions. This unit describes a method for creating libraries of chimeras through combinatorial assembly of gene fragments. It allows the experimenter to recombine genes of any identity and to select the sites where recombination takes place. Combinatorial recombination is achieved by generating gene fragments with specific overhangs, or sticky ends. The overhangs permit the fragments to be ligated in the correct order while allowing independent assortment of blocks with identical overhangs. Genes of any identity can be recombined so long as they share 3 to 5 base pairs of identity at the desired recombination sites. Simple adaptations of the method allow incorporation of specific gene fragments. Curr. Protoc. Protein Sci. 61:26.2.1‐26.2.20. © 2010 by John Wiley & Sons, Inc.

Keywords: chimera; recombination; directed evolution; protein design; library creation; combinatorial library

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Construction of a Combinatorial Gene Library
  • Alternate Protocol 1: Library Construction Using Synthetic Genes
  • Alternate Protocol 2: Library Construction to Incorporate Small (<40 bp) Blocks
  • Alternate Protocol 3: Library Construction to Recombine More than 8 Blocks
  • Alternate Protocol 4: Library Construction Using a Subset of Gene Fragments
  • Alternate Protocol 5: Chimera Library Construction
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction of a Combinatorial Gene Library

  Materials
  • Custom‐synthesized oligonucleotides for PCR primers
  • Gene sequences for proteins to be recombined
  • High‐fidelity DNA polymerase for PCR, e.g., Phusion
  • DNA gel extraction kit (Zymogen Zymoclean Gel DNA Recovery Kit or Qiagen Qiaquick Gel Extraction Kit)
  • PstI restriction endonuclease
  • SalI restriction endonuclease
  • DNA purification kit (Zymogen DNA Clean and Concentrator or Qiagen Qiaquick PCR Purification Kit)
  • Plasmid for construction (e.g., pBS KS+; Stratagene)
  • Calf alkaline phosphatase (Tabor, )
  • BsaXI restriction endonuclease
  • T4 DNA ligase (Tabor, )
  • NdeI restriction endonuclease
  • 150 × 15–mm LB agar plates ( appendix 4A) with antibiotic for transformation
  • SapI restriction endonuclease
  • BamHI restriction endonuclease
  • HindIII restriction endonuclease
  • Plasmid for final library cloning and expression (e.g., pET28)
  • 16°C water bath
  • Additional reagents and equipment for polymerase chain reaction ( appendix 4J), agarose gel electrophoresis ( appendix 4F), restriction enzyme digestion ( appendix 4I), introduction of plasmid DNA into cells ( appendix 4D), preparation of plasmid DNA ( appendix 4C), DNA sequencing (Ausubel et al., , Chapter 7), and quantitation of nucleic acids with absorption spectroscopy ( appendix 4K)
NOTE: All restriction enzymes mentioned in this protocol are examples, and may be replaced with other restriction enzymes compatible with the design of the experiment. SapI and BsaXI cleave outside their recognition sequences and should only be replaced with enzymes that have similar properties.

Alternate Protocol 1: Library Construction Using Synthetic Genes

  • Tag‐inserted genes made by a gene synthesis company, (e.g., DNA 2.0, https://www.dna20.com/)

Alternate Protocol 2: Library Construction to Incorporate Small (<40 bp) Blocks

  • T4 polynucleotide kinase (Tabor, )
  • Kinase buffer with 1 mM ATP
  • Thermal cycler capable of 1°C/sec ramp
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Figures

Videos

Literature Cited

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   Heinzelman, P., Snow, C.D., Wu, I., Nguyen, C., Villalobos, A., Govindarajan, S., Minshull, J., and Arnold, F.H. 2009. A family of thermostable fungal cellulases created by structure‐guided recombination. Proc. Natl. Acad. Sci. U.S.A. 106:5610‐5615.
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   Meinhold, P.M., Joern, J.M., and Silberg, J.J. 2003. Analysis of shuffled libraries by oligonucleotide probe hybridization. In Methods in Molecular Biology Vol. 231: Directed Evolution Library Creation (F. H. Arnold and G. Georgiou, eds.) pp. 177‐188. Humana Press, Totowa, New Jersey.
   Meyer, M.M., Silberg, J.J., Voigt, C.A., Endelman, J.B., Mayo, S.L., Wang, Z.‐G., and Arnold, F.H. 2003. Library analysis of SCHEMA‐guided recombination. Protein Sci. 12:1686‐1693.
   Nicot, C., Relat, J., Woldegiorgis, G., Haro, D., and Marrerro, P.F. 2002. Pig liver carnitine palmitoyltransferase. Chimera studies show that both the N‐ and C‐terminal regions of the enzyme are important for the unusual high malonyl‐CoA sensitivity. J. Biol. Chem. 277:10044‐10049.
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   Otey, C.R., Silberg, J.J., Endelman, J.B., Bandara, G., and Arnold, F.H. 2004. Functional evolution and structural conservation in chimeric cytochromes P450: Calibrating a structure‐guided approach. Chem. Biol. 11:309‐318.
   Roman, L.J., McLain, J., and Masters, B.S.S. 2003. Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitric‐oxide synthase reductase domain reveal structural and functional differences. J. Biol. Chem. 278:25700‐26707.
   Saraf, M.C., Horswill, A.R., Benkovic, S.J., and Maranas, C.D. 2004. FamClash: A method for ranking the activity of engineered enzymes. Proc. Natl. Acad. Sci. U.S.A. 101:4142‐4147.
   Saraf, M.C., Gupta, A., and Maranas, C.D. 2005. Design of combinatorial protein libraries of optimal size. Proteins 60:769‐777.
   Sieber, V., Martinez, C.A., and Arnold, F.H. 2001. Libraries of hybrid proteins from distantly related sequences. Nat. Biotechnol. 19:456‐460.
   Silberg, J.J., Endelman, J.B., and Arnold, F.H. 2004. SCHEMA‐guided protein recombination. In Methods in Enzymology Vol. 228 Protein Engineering (D.E. Robinson and J.P. Noel, eds.) pp. 35‐41. Elsevier Academic Press, Amsterdam.
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Internet Resources
  http://www.che.caltech.edu/groups/fha/
  Web site that supplies RASPP code for determining optimal recombination points.
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