Directed Evolution of Proteins In Vitro Using Compartmentalization in Emulsions

Eric A. Davidson1, Paulina J. Dlugosz1, Matthew Levy2, Andrew D. Ellington1

1 University of Texas at Austin, Austin, Texas, 2 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 24.6
DOI:  10.1002/0471142727.mb2406s87
Online Posting Date:  July, 2009
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Abstract

This unit describes a protocol for the directed evolution of proteins utilizing in vitro compartmentalization. This method uses a large number of independent in vitro transcription and translation (IVTT) reactions in water droplets suspended in an oil emulsion to enable selection of proteins that bind a target molecule. Protein variants that bind the target also bind to and allow recovery of the genes that encoded them. This protocol serves as a basis for carrying out selections in emulsions, and can potentially be modified to select for other functionalities, including catalysis. This selection method is advantageous compared to alternative selection protocols due to the ability to screen through very large‐size libraries and the ability to express and screen or select for functions that would otherwise be toxic or inaccessible to in vivo selections and screens. Curr. Protoc. Mol. Biol. 87:24.6.1‐24.6.12. © 2009 by John Wiley & Sons, Inc.

Keywords: directed evolution; in vitro compartmentalization; emulsion

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • DNA of interest
  • Mineral oil (molecular biology grade, RNase‐, DNase‐, protease‐free; Sigma, cat. no. M5904)
  • Span‐80 (sorbitane monooleate; e.g., Sigma, cat. no. S6760, or Fluka, cat. no. 85548)
  • Tween‐80
  • Triton X‐100
  • Cell‐free transcription and translation system, e.g., Roche RTS 100 E. coli HY Kit including:
    • E. coli lysate
    • Reaction mix
    • Amino acid mixture without methionine
    • Methionine
    • Reconstitution buffer
  • Tris‐buffered saline (TBS; appendix 22)
  • Quenching agent, e.g., 100 µM D‐biotin (Sigma‐Aldrich, cat. no. 47868) in TBS
  • Diethyl ether, H 2O‐saturated
  • Tris‐buffered saline/Tween 20 (TTBS; appendix 22)
  • Anti‐polyhistidine antibody bound to agarose beads (Sigma, cat. no. A5713)
  • Elution buffer (see recipe)
  • 95 × 16.8 mm polypropylene (13‐ml) Sarstedt tubes
  • 1.5 and 2‐ml microcentrifuge tubes
  • Spinplus 9.5 × 9.5 mm Teflon stir bars (VWR Scientific)
  • Stir plate (Corning Stirrer/Hot Plate PC‐420)
  • 90 × 50–mm (or similarly sized) glass beaker (to hold the test tube containing the emulsion)
  • Positive‐displacement pipettors (e.g., Microman from Gilson)
  • 30°C water bath
  • End‐over‐end rotator
  • Additional reagents and equipment for ethanol precipitation of DNA (unit 2.1), the polymerase chain reaction (PCR; unit 15.1), real‐time PCR (optional; unit 15.8), and agarose gel purification of DNA (unit 2.6)
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Figures

Videos

Literature Cited

Literature Cited
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