Elastin‐Like Polypeptides as a Purification Tag for Recombinant Proteins

Wafa Hassouneh1, Trine Christensen1, Ashutosh Chilkoti1

1 Duke University, Durham, North Carolina
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.11
DOI:  10.1002/0471140864.ps0611s61
Online Posting Date:  August, 2010
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Abstract

This unit presents a recombinant protein purification method that employs an elastin‐like polypeptide (ELP) as a purification tag. ELPs undergo a sharp and reversible phase transition when heated above their lower critical solution temperature. ELPs retain this behavior when they are fused to a protein, and thereby provide a simple method to isolate a recombinant ELP fusion protein from cell contaminants by cycling the solution through the insoluble and soluble phase of the ELP fusion protein using a procedure that is termed Inverse Transition Cycling. This method does not require the use of chromatography, so it is cost‐effective, easy to scale up, and easy to multiplex. Curr. Protoc. Protein Sci. 61:6.11.1‐6.11.16. © 2010 by John Wiley & Sons, Inc.

Keywords: Elastin‐like polypeptides (ELP); inverse transition cycling (ITC); purification tag; recombinant protein; non‐chromatographic purification; protein fusion; transition temperature

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Purification of Well Expressed ELP Fusion Proteins by Inverse Transition Cycling (ITC)
  • Alternate Protocol 1: Purification of Poorly Expressed ELP Fusion Proteins by Inverse Transition Cycling (ITC)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Well Expressed ELP Fusion Proteins by Inverse Transition Cycling (ITC)

  Materials
  • BL21(DE3) E. coli (Invitrogen) or BLR(DE3) E. coli (EMD Chemicals; http://www.emdchemicals.com/) competent cells transformed ( appendix 4D) with protein‐ELP fusion construct (see )
  • Ampicillin
  • Kanamycin
  • Terrific Broth (TB; appendix 4A), autoclaved
  • Ice/ethanol bath (in beaker)
  • 10% (v/v) polyethyleneimine (PEI)
  • NaCl (or other, stronger kosmotrope such as sodium citrate or ammonium sulfate)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Saturated NaCl solution in PBS or H 2O
  • 0.5 M CuCl 2 in H 2O (filtered)
  • Biotinylated protease corresponding to genetically engineered cut site (see text over step 13 below): e.g., biotinylated thrombin or TEV protease
  • Streptavidin‐agarose beads (Invitrogen, cat. no. SA100‐04)
  • 200‐ml and 4‐liter Erlenmeyer flasks
  • Shaking incubator
  • Low‐speed floor model temperature‐controlled centrifuge
  • Probe sonicator
  • 50‐ml centrifuge tubes
  • Temperature‐controlled high‐speed centrifuge with rotor accommodating 50‐ml tubes
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (unit 10.1), Coomassie Blue staining of gels (unit 10.5), and dialysis or ultrafiltration (unit 4.4)

Alternate Protocol 1: Purification of Poorly Expressed ELP Fusion Proteins by Inverse Transition Cycling (ITC)

  • Free ELP (composed of the same guest residues and molecular weight as the ELP tag in the ELP fusion protein (see annotation to step 2, below)
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Figures

Videos

Literature Cited

Literature Cited
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