Purification of Recombinant Proteins from Cultured Mammalian Cells by HaloTag Technology

Rachel Friedman Ohana1, Robin Hurst1

1 Promega Corporation, Madison, Wisconsin
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.31
DOI:  10.1002/0471142727.mb1031s110
Online Posting Date:  April, 2015
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Abstract

Cultured mammalian cells provide an environment ideal for producing functional recombinant mammalian proteins. However, low expression levels of recombinant proteins present a challenge for their detection and purification. This unit will focus on HaloTag, a protein fusion tag designed to bind selectively and covalently to a chloroalkane ligand that may be attached to a variety of functional groups, allowing both protein detection and immobilization. Detection of HaloTag‐fusion protein is achieved through binding to a fluorescent chloroalkane ligand, enabling rapid optimization of expression levels. HaloTag‐based purification uses covalent capture of the HaloTag fusion onto HaloLink resin coupled with proteolytic cleavage to release the protein of interest from the resin. Covalent binding provides efficient protein capture regardless of expression level and eliminates protein loss during washes of the resin and as a result, offers significant improvements in protein recovery and purity over traditional non‐covalent approaches. © 2015 by John Wiley & Sons, Inc.

Keywords: HaloTag; protein purification; fusion tag; fluorescent detection

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Transient Expression of HaloTag‐Fusion Protein in T150 Flasks
  • Alternate Protocol 1: Transient Expression of HaloTag‐Fusion Protein in Spinner Flasks
  • Basic Protocol 2: HaloTag‐Based Protein Purification from 120‐ml Culture
  • Support Protocol 1: Labeling and Detection of HaloTag‐Fusion Protein
  • Support Protocol 2: Optimization of Transfection and Expression Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transient Expression of HaloTag‐Fusion Protein in T150 Flasks

  Materials
  • HEK293/T cells
  • Polyethylenimine (PEI; 1 mg/ml; see recipe)
  • HaloTag fusion DNA construct
  • Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum (FetalClone I, HyClone)
  • Serum‐free DMEM (SF‐DMEM)
  • Phosphate‐buffered saline (PBS)
  • Equipment required for cell culture: T150 (TPP tissue culture flasks), CO 2 incubator and tissue culture cell scraper (for scraping adherent cells)
  • Tube shaker (Eppendorf, Thermomixer or equivalent)
  • 15‐ml centrifuge conical tubes
  • Cell counting equipment: microscope, hemocytometer and 0.4% trypan blue for cell staining or equivalent ( appendix 3F)
  • Centrifuge with swinging bucket

Alternate Protocol 1: Transient Expression of HaloTag‐Fusion Protein in Spinner Flasks

  Additional Materials (also see protocol 1)
  • Equipment required for cell culture: disposable spinner flasks (125 ml, Corning)
  • 100× Pluronic F‐68 polyol

Basic Protocol 2: HaloTag‐Based Protein Purification from 120‐ml Culture

  Materials
  • Harvested cells or collected medium (secreted proteins) from a 120‐ml culture expressing HaloTag‐fusion protein
  • Purification buffer (see recipe)
  • HaloTag mammalian detection and purification system (Promega; system includes HaloLink resin, HaloTEV protease, protease inhibitor cocktail, spin columns and HaloTag TMRDirect ligand)
  • 15‐ml centrifuge conical tubes
  • 1.5‐ml microcentrifuge tubes
  • Probe sonicator with microtip
  • End‐over‐end tube rotator and/or shaking platform (to ensure proper mixing of the resin)
  • Centrifuge with swinging bucket
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2, Gallagher, ), Coomassie blue staining of protein gels (unit 10.6, Sasse and Gallagher, ), and Bradford protein assay (unit 10.1, Simonian and Smith, )

Support Protocol 1: Labeling and Detection of HaloTag‐Fusion Protein

  Materials
  • Cell lysate (from culture expressing a HaloTag‐fusion protein) or cell medium containing a secreted HaloTag‐fusion protein ( protocol 3; binding the HaloTag‐fusion protein to the HaloLink resin, step 1)
  • Unbound fraction ( protocol 3; binding the HaloTag‐fusion protein to the HaloLink resin, step 3)
  • HaloTag TMRDirect ligand (excitation 532 nm, emission 580 nm)
  • Dimethyl sulfoxide (DMSO)
  • Aluminum foil or amber tube (for storage of working solution)
  • Fluorescent imager (Typhoon 9410, GE Healthcare or equivalent)
  • Heating block (Thermomixer, Eppendorf, or equivalent)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2, Gallagher, )

Support Protocol 2: Optimization of Transfection and Expression Conditions

  Materials
  • HEK293/T cells
  • Polyethylenimine (PEI, 1 mg/ml; see recipe)
  • HaloTag‐fusion construct
  • Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum (FetalClone I, HyClone)
  • Serum‐free Dulbecco's modified Eagle's medium (SF‐DMEM)
  • HaloTag TMRDirect ligand (excitation 532 nm, emission 580 nm)
  • Detergent lysis buffer (see recipe)
  • Equipment required for cell culture (see protocol 1)
  • Cell counting equipment: microscope, hemocytometer and 0.4% trypan blue for cell staining or equivalent ( appendix 3F)
  • Fluorescence imager (Typhoon 9410, GE Healthcare or equivalent)
  • 24‐well tissue culture plates
  • Tube shaker (Thermomixer, Eppendorf, or equivalent)
  • Heating block (Thermomixer, Eppendorf, or equivalent)
  • Plate shaker
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2, Gallagher, )
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Figures

Videos

Literature Cited

Literature Cited
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