Expression and Purification of ZEBRA Fusion Proteins and Applications for the Delivery of Macromolecules into Mammalian Cells

Romy Rothe1, Jean‐Luc Lenormand1

1 HumProTher Laboratory, TheREx‐GREPI, University of Joseph Fourier, UFR de Médecine, La Tronche, France
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.11
DOI:  10.1002/0471140864.ps1811s54
Online Posting Date:  November, 2008
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Abstract

The recent development of peptide carriers for efficient and specific delivery of biologically active molecules into mammalian cells represents a major advance in the study of both normal and uncontrolled cell growth. In the past few years, this technology has been successfully applied to the delivery of therapeutic molecules in animal models, and now some of these carriers are available in the clinic for the treatment of some human diseases. This unit describes the production, in a bacterial expression system, of reporter proteins (EGFP and β‐galactosidase) fused to a transduction domain of the Epstein‐Barr virus ZEBRA protein, as well as purification of the fusion proteins. The purified fusion proteins can be added to any of a large spectrum of mammalian cells and the internalization process measured by flow cytometry and fluorescence microscopy on live cells. Fluorescence microscopy on fixed cells is used to study their intracellular distribution. Curr. Protoc. Protein Sci. 54:18.11.1‐18.11.29. © 2008 by John Wiley & Sons, Inc.

Keywords: protein expression; fusion protein; protein delivery; endocytosis; intracellular distribution; therapeutic proteins

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Construction of Zebra Fusion DNA Using a pET15b VECTOR
  • Basic Protocol 2: Expression of Recombinant Zebra Fusion Proteins
  • Basic Protocol 3: Batch and Large‐Scale Purification of Recombinant Zebra Fusion Proteins by Affinity Chromatography
  • Basic Protocol 4: Electrophoretic Mobility Shift Assay (EMSA) to Monitor AP‐1 Probe Binding by ZEBRA Fusion Proteins
  • Basic Protocol 5: Cellular Transduction Assays Using ZEBRA Fusion Proteins
  • Basic Protocol 6: Analysis of Intracellular ZEBRA Fusion Protein Localization
  • Support Protocol 1: Fluorescent Labeling of Truncated ZEBRA Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction of Zebra Fusion DNA Using a pET15b VECTOR

  Materials
  • 10× PCR buffer ( appendix 4J)
  • 10 mM dNTP Mix (Roche Applied Science)
  • Template DNA
  • 25 mM MgCl 2
  • Proofreading DNA polymerase: e.g., PfuUltra High‐Fidelity DNA Polymerase (Stratagene) or Isis Proofreading DNA Polymerase (QBiogene)
  • 5× loading buffer ( appendix 4F)
  • 0.8% to 2% agarose gel
  • Ethidium bromide (Interchim)
  • QIAquick Gel Extraction kit (QIAGEN)
  • pET15b plasmid (Novagen EMD Biosciences; Fig. )
  • Restriction enzymes NdeI and XhoI
  • 50× TAE‐buffer (e.g., Sigma; also see appendix 4F)
  • MassRuler, DNA ladder (Fermentas)
  • Rapid DNA ligation kit (Roche Applied Science)
  • Library Efficiency DH5α Chemically Competent cells (Invitrogen)
  • 10‐cm LB agar plates supplemented with ampicillin ( appendix 4A)
  • QIAprep Spin Miniprep kit (QIAGEN)
  • 80% sterile glycerol
  • Quickchange Site‐Directed Mutagenesis kit (Stratagene)
  • T7 primer (AGOWA Sequencing Service)
  • Mastercycler Gradient (Eppendorf)
  • UV table (VWR)
  • 37°C incubator
  • 14‐ml round‐bottom tubes (BD Falcon)
  • Shaking platform
  • Sterile screw‐cap microcentrifuge tube
  • Vortex
  • Additional reagents and equipment for PCR ( appendix 4J) and electrophoresis ( appendix 4F)

Basic Protocol 2: Expression of Recombinant Zebra Fusion Proteins

  Materials
  • Glycerol culture of transformed E. coli bearing pET15b vector containing ZEBRA fusion construct ( protocol 1)
  • E. coli BL21 (DE3) competent cells (Novagen EMD Biosciences)
  • E. coli Origami (DE3) competent cells (Novagen EMD Biosciences)
  • E. coli Rosetta (DE3) competent cells (Novagen EMD Biosciences)
  • E. coli BL21‐CodonPlus (DE3)‐RIPL competent cells (Stratagene)
  • LB‐agar plates supplemented with appropriate antibiotics ( appendix 4A)
  • Luria broth (LB medium; Sigma)
  • 10 mg/ml ampicillin (Sigma)
  • 1 M stock solution of isopropyl‐1‐thio‐β‐D‐galactosidase (IPTG; MP biomedicals Qbiogen)
  • Buffer A (see recipe)
  • Complete Protease Inhibitor cocktail tablets, EDTA‐free (1 tablet per 50 ml buffer; Roche Applied Science)
  • Complete Protease Inhibitor cocktail tablets, Mini EDTA‐free (1 tablet per 10 ml buffer; Roche Applied Science)
  • Protease inhibitors (Pepstatin, E‐64, Aprotinin, Pefabloc; Roche Applied Science)
  • Buffer B (see recipe)
  • Lysis/binding buffer (see recipe)
  • 37°C incubator
  • Inoculating loop or sterile toothpick
  • 14‐ml round‐bottom tubes (BD Falcon)
  • 37°C shaking incubator
  • Refrigerated centrifuge (e.g., Sigma)
  • Sonicator with a microtip probe (Branson Sonic Power)
  • 0.45‐µm nitrocellulose membrane (BioRad)
  • Additional reagents and equipment for introducing plasmid DNA into cells ( appendix 4D) and SDS‐PAGE (unit 10.1)

Basic Protocol 3: Batch and Large‐Scale Purification of Recombinant Zebra Fusion Proteins by Affinity Chromatography

  Materials
  • Bacterial pellet from 3‐ml overnight culture containing ZEBRA fusion protein ( protocol 2)
  • Lysis/binding buffer (see recipe)
  • MagnaHis beads purification system (Roche Applied Science)
  • Washing buffers (see reciperecipes)
  • Elution buffer (see recipe)
  • SDS‐PAGE gel (Criterion XT Bis‐Tris Gel, 4% to 12%, 18‐well, 30 µl, BioRad or Criterion XT Bis‐Tris Gel, 4‐12%, 26‐well, 15 µl, BioRad) or regular polyacrylamide gel
  • DNase I, recombinant (Roche Applied Science)
  • Protease inhibitors: pepstatin, E‐64, aprotinin, pefabloc (Roche Applied Science)
  • Sonicator (Branson Sonic Power)
  • Centrifuge (Sigma 3‐18K)
  • 1.5‐ml microcentrifuge tube
  • Magnetic stand for up to 12 microcentrifuge tubes (Promega)
  • End‐over‐end rotator
  • HisGraviTrap columns (Amersham GE Healthcare)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and analyzing gels using Coomassie blue staining (unit 10.5) and immunoblotting (unit 10.10)
NOTE: All buffers must be filtered and cooled on ice before use. Add the protease inhibitors just before use.

Basic Protocol 4: Electrophoretic Mobility Shift Assay (EMSA) to Monitor AP‐1 Probe Binding by ZEBRA Fusion Proteins

  Materials
  • Acrylamide mix (29:1; acrylamide:bisacrylamide, Sigma Aldrich)
  • 10 × TBE (Tris‐borate/EDTA) buffer (see recipe)
  • AP‐1 oligonucleotides (designed according to AP‐1 nucleotide sequence):
    • 5′‐AGCACTGACTCATGAAGT‐3′
    • 5′‐TACTTCATGAGTCAGTGCT‐3′
  • Biotin dUTPs (Roche)
  • Klenow buffer (Roche)
  • Klenow polymerase (Roche)
  • 50 µg of purified ZEBRA minimal transduction domain fusion protein (from protocol 2)
  • Nushift kit reaction (Active Motif) containing:
    • Binding Buffer B‐1
    • Binding Buffer C‐1
    • Stabilizing Solution
  • 10 mM stock 1,4‐dithio‐DL‐threitol (DTT, Sigma)
  • Poly‐dI‐dC (Pierce)
  • 10× nondenaturating loading buffer
  • Transfer buffer (see recipe), for semidry transfer
  • LightShift Chemiluminescent EMSA Kit (Pierce, cat. no. 20148)
  • Saran wrap
  • Gel electrophoresis apparatus
  • Thermal cycler
  • Microspin G‐25 spin column (Active Motif)
  • Microcentrifuge tubes
  • Scalpel
  • Hybond‐N+ membrane (Amersham GE Healthcare)
  • Stratalinker UV Crosslinker (Stratagene)
  • Additional reagents and equipment for nondenaturing electrophoresis (unit 10.3) and transferring gel lanes to a membrane using a semidry transfer unit (unit 10.7)

Basic Protocol 5: Cellular Transduction Assays Using ZEBRA Fusion Proteins

  Materials
  • 0.04% trypan blue solution (Invitrogen)
  • HeLa cell lines (ATCC: CCL‐2)
  • Saos‐2 cell lines (ATCC: HTB‐85)
  • Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
  • McCoy's 5A medium (Invitrogen)
  • Fetal bovine serum (FBS; Invitrogen)
  • 2 mM L‐glutamine (Invitrogen)
  • Penicillin/streptomycin (Invitrogen)
  • Sterile phosphate‐buffered saline (PBS; prepared from 10× stock; see recipe), 1×
  • Bovine serum albumin (BSA)
  • 0.5% trypsin/EDTA (Invitrogen)
  • Appropriate culture medium
  • Heparin (Sigma)
  • Propidium iodide
  • Inverted phase contrast microscope
  • Hemacytometer: modified Neubauer chamber
  • 12‐well plates (BD Falcon)
  • 4‐ and 8‐chamber slides, glass surface and 0.8‐cm2 area, Lab‐Tek glass slides (Nunc)
  • Slide‐A‐Lyzer Dialysis Cassette (Pierce)
  • Vivaspin columns (Sartorius), optional
  • Microcentrifuge tubes
  • Fluorescence‐activated cell sorter (FACScalibur; Becton Dickinson)
  • Fluorescence microscope (e.g., Nikon Eclipse TE2000‐E)
  • Additional reagents and equipment for determining cell number and viability with a hemacytometer and trypan blue staining ( appendix 3C), SDS‐PAGE (unit 10.1), and Coomassie blue staining (unit 10.5)

Basic Protocol 6: Analysis of Intracellular ZEBRA Fusion Protein Localization

  Materials
  • Target cells in 25‐ to 175‐cm2 culture vessels
  • Purified ZEBRA (e.g., ZEBRA‐EGFP or ZEBRA‐β‐gal) fusion protein
  • Phosphate‐buffered saline (PBS; prepared from 10× stock; see recipe), 1×
  • 0.5% trypsin/EDTA (Invitrogen)
  • Appropriate culture medium
  • Heparin (Sigma)
  • Propidium iodide
  • Aqueous mounting medium (Dako)
  • 4% (w/v) paraformaldehyde (PFA) in PBS
  • 0.1% Saponin
  • 5% BSA
  • Mouse Anti‐histidine antibody (clone 1DM‐1H7; Euromedex)
  • Secondary antibodies Anti‐mouse IgM Alexa Fluor 488 and Anti‐rabbit IgG Alexa Fluor 555 (Molecular Probes, Invitrogen)
  • Hoechst 33258 (Molecular Probes, Invitrogen)
  • β‐galactosidase staining kit (Sigma Aldrich) containing:
    • 10× fixation buffer
    • MgCl 2
    • Potassium ferricyanide
    • Potassium ferrocyanide
    • X‐Gal
  • 37°C incubator
  • Microcentrifuge tubes
  • Fluorescence‐Activated Cell Sorter (FACScalibur; Becton Dickinson)
  • 8‐well chamber slide (Nunc)
  • Fluorescence microscope
  • Zeiss LSM510 (NLO) laser confocal microscope
  • Inverted Microscope Nikon Eclipse TE2000‐E equipped with epifilters

Support Protocol 1: Fluorescent Labeling of Truncated ZEBRA Proteins

  Materials
  • Purified ZEBRA fusion protein ( protocol 2)
  • Sterile phosphate‐buffered saline (PBS; prepared from 10× stock; see recipe), 1×
  • Bovine serum albumin (BSA)
  • Slide‐A‐Lyzer Dialysis Cassette (Pierce)
  • Ultra‐Amicon concentration tubes (Millipore)
  • Alexa Fluor 647 Microscale Protein Labeling Kit (Molecular Probes, Invitrogen)
  • Alexa Fluor 488 protein labeling Kit (Roche Applied Science)
  • Fluorescence‐Activated Cell Sorter (FACScalibur; Becton Dickinson)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and Coomassie blue staining (unit 10.5)
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Figures

Videos

Literature Cited

Literature Cited
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