Protein Transduction: Generation of Full‐Length Transducible Proteins Using the TAT System

Michelle Becker‐Hapak1, Steven F. Dowdy2

1 Washington University School of Medicine, Saint Louis, Missouri, 2 University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 20.2
DOI:  10.1002/0471143030.cb2002s18
Online Posting Date:  May, 2003
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Abstract

This unit describes the technology that allows an investigator to transduce full‐length proteins by utilizing a minimal, eleven‐amino acid, HIV‐TAT transduction domain that can be fused to a protein of choice using the pTAT or pTAT‐HA protein expression plasmids. Bacterial expression, followed by solubilization of protein aggregates with a denaturing agent, affords high yields of transducible fusion protein. The fusion protein, once added to the culture medium, can cross the cell membrane and then be degraded or refolded by the cellular machinery. Correct targeting and function of the fusion protein can be easily examined by fluorescent microscopy or immunohistochemistry. This strategy was established and improved to its current state by the purification and transduction of a multitude of fusion proteins. Because the pool of fusion proteins spans many different functions, the protocols cover a wide variety of commonly used protein isolation and characterization methods.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Expression, Verification, and Yield Optimization of TAT‐Fusion Proteins
  • Basic Protocol 2: Large‐Scale Isolation of the TAT‐Fusion Protein
  • Alternate Protocol 1: Use of Ion‐Exchange Gravity Columns Instead of FPLC
  • Alternate Protocol 2: Direct Buffer Exchange of Urea‐Denaturated Protein
  • Alternate Protocol 3: Dialysis of the Urea‐Denaturated Protein
  • Alternate Protocol 4: Isolation of Soluble TAT‐Fusion Proteins
  • Basic Protocol 3: Transduction and Detection with Fluorophore‐Labeled Fusion Protein
  • Alternate Protocol 5: Transduction and Detection by Indirect Immunofluorescence
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Expression, Verification, and Yield Optimization of TAT‐Fusion Proteins

  Materials
  • Pure pTAT/pTAT‐HA expression vectors (Nagahara et al., ) with and without the gene of interest inserted (available from Dr. S. Dowdy, )
  • E. coli strains BL‐2 (DE3) pLysS (Novagen) and DH5α (Invitrogen, Life Technologies)
  • LB medium and plates both containing 50 µg/ml ampicillin (see recipe)
  • 2× SDS‐PAGE sample buffer ( appendix 2A)
  • Antibody specific for target protein or anti‐HA mAb (Berkeley Antibody Company) if using pTAT‐HA vector
  • Glycerol, ultrapure, 50% (v/v), sterile filtered
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), Coomassie blue staining (unit 6.6), and basic molecular biology procedures (including transformation of bacteria and IPTG induction; see appendix 3A)

Basic Protocol 2: Large‐Scale Isolation of the TAT‐Fusion Protein

  Materials
  • LB medium containing 50 µg/ml ampicillin (see recipe)
  • Glycerol stock of clone with high expression of TAT fusion protein (see protocol 1)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Buffer Z (see recipe) containing 1× protease inhibitors (see recipe)
  • 5 M (340 g/liter) imidazole (store in foil‐wrapped bottle at 4°C)
  • 50% (w/v) stock suspension of Ni‐NTA agarose (Qiagen)
  • 100 mM, 250 mM, 500 mM, and 1 M imidazole in recipebuffer Z (see recipe for buffer Z), prepared fresh daily
  • PBS ( appendix 2A) containing 0.1% (w/v) sodium azide
  • 20 mM HEPES, pH 8 (for Mono Q resin) or pH 6.5 (for Mono S resin)
  • Buffer A (binding): 20 mM HEPES, pH 8.0, for Mono Q; pH 6.5 for Mono S
  • Buffer B (elution): 20 mM HEPES/1 M NaCl, pH 8.0, for Mono Q; pH 6.5 for Mono S
  • PBS ( appendix 2A) containing 1× protease inhibitors (see recipe)
  • Bovine serum albumin (BSA)
  • Glycerol (ultrapure), 50% (v/v) sterile filtered
  • Sorvall refrigerated centrifuge with GSA rotor, or equivalent
  • Sonicator with microprobe (Branson)
  • Disposable 50‐ml Econo columns (Bio‐Rad)
  • Mono Q or Mono S 5/5 or 10/10 ion‐exchange FPLC columns or bulk resin (Resource Q or S), all products of Amersham Pharmacia Biotech
  • FPLC apparatus
  • PD‐10 gel filtration columns (Amersham Pharmacia Biotech)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and Coomassie blue staining (unit 6.6)

Alternate Protocol 1: Use of Ion‐Exchange Gravity Columns Instead of FPLC

  • 30‐µm Resource Q or S ion‐exchange resin (Amersham Pharmacia Biotech; see protocol 2 for choice of resin)
  • 50‐ml Econo columns (Bio‐Rad)
NOTE: Perform all steps at 4°C and add 1× protease inhibitors (see recipe) to all solutions.

Alternate Protocol 2: Direct Buffer Exchange of Urea‐Denaturated Protein

  • Serum‐free culture medium (e.g., RPMI‐1640) without antibiotics, containing 1× protease inhibitor cocktail (see recipe) or PBS plus recipe1× protease inhibitor cocktail

Alternate Protocol 3: Dialysis of the Urea‐Denaturated Protein

  • Slide‐A‐Lyzer dialysis cassettes (Pierce) with membrane of MWCO appropriate for protein of interest

Alternate Protocol 4: Isolation of Soluble TAT‐Fusion Proteins

  Materials
  • Fluorescein isothiocyanate (FITC; Molecular Probes)
  • DMSO
  • Purified fusion protein (see protocol 2 and Alternate Protocols protocol 31 to protocol 64)
  • 10× FITC conjugation buffer (see recipe)
  • PBS ( appendix 2A) containing 1× protease inhibitors (see recipe)
  • Glycerol, ultrapure
  • Cell line of interest for transduction or Jurkat T cell culture
  • Paraformaldehyde fix solution (see recipe)
  • Antifade mounting medium (Molecular Probes)
  • Clear nail polish
  • PD‐10 gel‐filtration columns (Amersham Pharmacia Biotech)
  • Microscope slides and coverslips

Basic Protocol 3: Transduction and Detection with Fluorophore‐Labeled Fusion Protein

  Materials
  • Adherent cells of interest for transduction, e.g. NIH 3T3 cells
  • Culture medium for NIH 3T3 cells (i.e., DMEM/10% FBS)
  • Purified fusion protein (see protocol 2 and Alternate Protocols protocol 31 to protocol 64)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold and room temperature
  • Paraformaldehyde fix solution (see recipe)
  • 100% ethanol, ice‐cold
  • 1% and 0.1% (w/v) bovine serum albumin (BSA) in PBS (prepare from 10% w/v BSA stock)
  • Primary antibody: antibody of choice to fusion protein or mAb to the HA epitope (Berkeley Antibody Company)
  • TRITC‐ or PE‐labeled secondary antibody
  • 0.2 µg/ml DAPI (prepare fresh from 1 mg/ml DAPI stock; store stock in dark at 4°C)
  • Slowfade mounting medium (Molecular Probes)
  • Clear nail polish
  • Lab‐Tek 8‐chamber glass slides with lids (Nalge Nunc International)
  • 40°C heat block
  • 50 × 24–mm coverslips (Fisher)
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Figures

Videos

Literature Cited

Literature Cited
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   Hall, D.J., Cui, J., Bates, M.E., Stout, B.A., Koenderman, L., Coffer, P.J., and Bertics, P.J. 2001. Transduction of a dominant‐negative H‐Ras into human eosinophils attenuates extracellular signal‐regulated kinase activation and interleukin‐5‐mediated cell viability. Blood 98:2014‐2021.
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   Ho, A., Schwarze, S.R., Mermelstein, S.J., Waksman, G., and Dowdy, S.F. 2001. Synthetic protein transduction domains: Enhanced transduction potential in vitro and in vivo. Cancer Res. 61:474‐477.
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Key References
   Amersham Biosciences, 2002. See above.
  Excellent resource for the theory of ion‐exchange chromatography and troubleshooting measures for difficulties associated with using strong ion‐exchange resins.
   Backus et al., 2001. See above.
  Addresses all safety issues regarding the safe handling procedures for individuals working with TAT fusion proteins.
   Nagahara et al., 1998. See above.
  First description of the utility of TAT‐fused full‐length fusion proteins.
   Schwarze et al., 1999. See above.
  First demonstration in vivo delivery of a biologically active TAT‐fusion protein and showed that TAT‐fusions as large as 120 kDa could be delivered to virtually every cell type of a live mouse.
   Qiagen. 1997. The QIAexpressionist: A Handbook for high level expression of 6× His‐tagged proteins. Qiagen, Chatsworth, Calif.
  Contains general procedures and considerations for the expression and purification of b‐His‐tagged proteins.
   Wadia and Dowdy, 2002. See above.
  Recent review on protein transduction methodologies.
Internet Resources
  http://www.expasy.ch/tools/pi_tool.html
  Tools for rapid calculation of pI of a protein.
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