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Directed Evolution of Proteins In Vitro Using Compartmentalization in Emulsions

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

¹University of Texas at Austin, Austin, Texas
²Albert 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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Basic Protocol
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol

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 2)
Quenching agent, e.g., 100 µM d-biotin (Sigma-Aldrich, cat. no. 47868) in TBS
Diethyl ether, H₂O-saturated
Tris-buffered saline/Tween 20 (TTBS; appendix 2)
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 2.1A), 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

Figure 24.6.1

Scheme for binding selections in in vitro compartments. (A) A generic template for binding selections (top), and the template for streptavidin selections (bottom) as further described in Levy and Ellington (2008). The leftmost triangle represents the target molecule attached to the template (e.g., biotin). The promoter (T7 RNA polymerase promoter) is required for transcription initiation while the ribosome binding site (RBS) enhances translation initiation. The “tag” is part of the protein sequence (a hexahistidine or His₆ tag in the current example) and enables affinity purification of the translated protein. (B) Selection schema showing recovery of a desired template and protein (light gray) and removal of inactive template (dark gray). From top to bottom: Compartments are formed containing no more than 1 gene. The templates are transcribed and translated to produce proteins. Some proteins will bind the target molecule conjugated to their templates. The translated proteins must retain their templates throughout the recovery and wash process. While nonbinding proteins will also be captured, they will not carry their corresponding templates with them. Captured templates will be amplified by PCR and used in subsequent rounds of selection.

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Literature Cited

Literature Cited
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