Recombinant Protein Purification by Self‐Cleaving Elastin‐like Polypeptide Fusion Tag

Wan‐Yi Wu1, Baley A. Fong1, Allison G. Gilles1, David W. Wood1

1 Princeton University, Princeton, New Jersey
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 26.4
DOI:  10.1002/0471140864.ps2604s58
Online Posting Date:  November, 2009
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Abstract

This unit presents a rapid and simple method for the nonchromatographic purification of recombinant proteins expressed in E. coli. This method relies on a thermally responsive elastin‐like polypeptide (ELP) tag, where the tagged protein is precipitated using a mild temperature shift. The tag is then induced to self‐cleave by a mild pH shift and is subsequently removed by a final thermal precipitation. The result is a purified native protein target, without the requirement for affinity apparatus or protease removal of the tag. This protocol describes the required cloning methods to insert a given target into the expression vector, as well as the general method for purifying the resulting expressed protein. Curr. Protoc. Protein Sci. 58:26.4.1‐26.4.18. © 2009 by John Wiley & Sons, Inc.

Keywords: intein; elastin‐like polypeptide; protein purification; Gateway cloning; nonchromatographic bioseparations

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cloning a Target Protein Gene into the pET/EI‐X Plasmid Via Conventional Methods to Yield an ELP‐Intein‐Target Protein Expression Plasmid
  • Alternate Protocol 1: Inserting a Target Protein Gene into the Tag‐I‐X Expression Vector Via the Gateway Cloning Method
  • Basic Protocol 2: ELP‐Tagged Target Protein Expression and Purification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cloning a Target Protein Gene into the pET/EI‐X Plasmid Via Conventional Methods to Yield an ELP‐Intein‐Target Protein Expression Plasmid

  Materials
  • pMAL‐c2x (example template for PCR reaction of MBP target gene protein; New England Biolabs)
  • 10× Ex Taq polymerase buffer (Takara Bio)
  • 2 mM dNTP mix (New England Biolabs)
  • 5′ and 3′ primers (see )
  • Taq polymerase (TakaRa Bio)
  • QIAquick PCR Purification Kit (Qiagen)
  • pET/EI‐X plasmid (Available from the authors, please contact for related material transfer agreement documents)
  • 10× NEB 2 buffer (New England Biolabs)
  • 10× BSA (New England Biolabs)
  • Restriction enzymes BsrGI and HindIII (New England Biolabs)
  • Agarose (Invitrogen)
  • QIAquick Gel Extraction Kit (Qiagen)
  • 10× T4 DNA ligase buffer (New England Biolabs)
  • T4 DNA ligase (New England Biolabs)
  • DH5α competent cells (F, φ80dlacZΔM15, Δ(lacZYA‐argF)U169, deoR, recA1, endA1, hsdR17(rk, mk+), phoA, supE44, λ, thi‐1, gyrA96, relA1)
  • LB + 100 µg/ml ampicillin agar plate (see recipe)
  • Ampicillin (see recipe)
  • QIAprep Spin Miniprep Kit (Qiagen)
  • Thermal cycler
  • 0.2‐ml PCR tubes
  • 37° and 16°C incubators
  • Additional reagents and equipment for separating the digested DNA fragments on 1% agarose gel ( appendix 4F)
NOTE: The DH5α strain is available from several sources, including Invitrogen (www.invitrogen.com), and it can be purchased as prepared competent cells, or made competent using a variety of published protocols (e.g. calcium competent or electrocompetent). In our laboratory, we typically make the cells Z‐competent by using the Z‐competent transformation buffer kit purchased from Zymo Research (www.zymoresearch.com). Regardless of whether competent cells are purchased or prepared, the transformations should be carried out according to the associated protocols for that type of competent cells.

Alternate Protocol 1: Inserting a Target Protein Gene into the Tag‐I‐X Expression Vector Via the Gateway Cloning Method

  Materials
  • Entry vector pENTRΔI G(C)ccdB‐CM (available from authors, as described in Gillies et al., )
  • Target protein gene (see protocol 1)
  • 10× NEB 2 buffer (New England Biolabs)
  • 10× BSA (New England Biolabs)
  • Restriction enzymes BsrGI, BsiWI, and HindIII (New England Biolabs)
  • QIAquick Gel Extraction Kit (Qiagen)
  • T4 DNA ligase (New England Biolabs)
  • 10× T4 DNA ligase buffer (New England Biolabs)
  • DH5α competent cells (F, φ80dlacZΔM15, Δ(lacZYA‐argF)U169, deoR, recA1, endA1, hsdR17(rk, mk+), phoA, supE44, λ, thi‐1, gyrA96, relA1)
  • LB + 50 µg/ml kanamycin plate (see recipe)
  • QIAprep Spin Miniprep Kit (Qiagen)
  • Destination vectors pDEST/CΔI G(N), pDEST/EΔI G(N), pDEST/MΔI G(N) and pDEST/PPPΔI G(N) (available from authors, as described in Gillies et al., )
  • TE buffer ( appendix 2E), pH 8.0
  • LR clonase II enzyme mix (Invitrogen)
  • Proteinase K (Invitrogen)
  • LB + 100 µg/ml ampicillin plate (see recipe)
  • 37°, 16°, and 25°C incubators
  • Vortex
  • Additional reagents and equipment for generating the target gene ( protocol 1), separating the digested DNA fragments on a 1% agarose gel ( appendix 4F), and verifying restricted colonies by restriction digest ( appendix 4I)

Basic Protocol 2: ELP‐Tagged Target Protein Expression and Purification

  Materials
  • pET/EI‐MBP or pDEST/EI‐M (expression plasmid; see protocol 1 or the protocol 2 for plasmid construction protocols; the plasmids generated in these protocols are the expression plasmids)
  • BLR competent cells [FompT hsdS B (r Bm B) gal dcm (DE3) Δ(srl‐recA)306::Tn10 (TetR); Novagen]
  • LB agar plates supplemented with 100 µg/ml ampicillin (see recipe)
  • LB medium (see recipe)
  • Ampicillin (see recipe)
  • Terrific broth (see recipe)
  • Lysis buffer (see recipe)
  • Dry ice or liquid nitrogen, optional
  • 3 M NaCl
  • Cleaving buffer (see recipe)
  • 37°C shaking incubator
  • Glass culture tube (o.d × length = 20 × 150–mm)
  • Centrifuge
  • −20°C freezer
  • Sonicator
  • Vortex
  • Additional reagents and equipment for analyzing the fractions by SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
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