Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells

Irene Nigi1, Louise Fairall1, John W.R. Schwabe1

1 Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.28
DOI:  10.1002/cpps.44
Online Posting Date:  November, 2017
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Abstract

Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies. Mammalian expression systems can often produce natively folded proteins, sometimes with native post‚Äźtranslational modifications. However, such expression systems are underutilized due to the perception that they are costly, technically challenging and result in limited protein yields. In fact, HEK 293F cells are straightforward to grow, transfect with high efficiency and often produce significant yields of recombinant proteins. In this unit, we describe a method to express and purify milligram quantities of a human protein complex from HEK 293F cells grown in suspension transiently transfected with the appropriate plasmids. ¬© 2017 by John Wiley & Sons, Inc.

Keywords: cell culture; mammalian cells; HEK 293F; transient transfection; protein complexes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Large‐Scale Transient Transfection
  • Support Protocol 1: Large‐Scale Plasmid Preparation
  • Basic Protocol 2: Protein Complex Purification
  • Alternate Protocol 1: Purification of Protein Complexes Using Sucrose Gradients
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Large‐Scale Transient Transfection

  Materials
  • HEK 293Freestyle suspension‐adapted cells (Gibco)
  • Serum‐free FreeStyle 293 expression medium at 37°C (stored at 4°C) (Gibco)
  • Filter‐sterilized DNA (from the protocol 2Support Protocol)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • Polyethylenimine solution (PEI; see recipe)
  • Roller bottle, 490 cm3 (Corning) with vented cap (Corning)
  • Orbital shaker incubator, 37°C, 120 rpm, 5% to 8% CO 2
  • Hemacytometer
  • Vortex mixer
  • Centrifuge

Support Protocol 1: Large‐Scale Plasmid Preparation

  Materials
  • 2TY medium (see recipe)
  • Starting buffer (see recipe)
  • Lysozyme buffer (see recipe)
  • Alkaline buffer (see recipe)
  • Neutralization buffer (see recipe)
  • Isopropanol
  • Resuspension buffer 1 (see recipe)
  • 5 M lithium chloride
  • Ethanol
  • Resuspension buffer 2 (see recipe)
  • Heat‐treated Ribonuclease A from bovine pancreas (Sigma‐Aldrich)
  • PEG buffer (see recipe)
  • Chloroform
  • 5 M NaCl
  • 70% ethanol
  • TE buffer (see recipe)
  • Shaking incubator
  • Centrifuge
  • 50‐ml centrifuge tubes
  • Miracloth (22‐25 μm pore size; Millipore)
  • 0.22‐μm syringe filters
  • Vortex mixer

Basic Protocol 2: Protein Complex Purification

  Materials
  • Cell pellet ( protocol 1)
  • Protein lysis buffer 1 (see recipe)
  • anti‐FLAG® M2 affinity gel (Sigma Aldrich)
  • Wash buffer (see recipe)
  • TEV cleavage buffer (see recipe)
  • Protein loading buffer (2× see recipe)
  • Tobacco Etch Virus (TEV) protease
  • Gel filtration buffer (see recipe)
  • SDS PAGE
  • Coomassie stain
  • Glass homogenizer
  • Sonicator
  • Centrifuge
  • 50‐ and 15‐ml centrifuge tubes
  • Amicon ® Ultra centrifugal concentrator (Merck Millipore), 10 K molecular‐weight cut‐off
  • 0.22‐μm centrifugal filter (Merck Millipore)
  • Superose 6 (10/300) GL column (GE Healthcare)

Alternate Protocol 1: Purification of Protein Complexes Using Sucrose Gradients

  Materials
  • Protein sample (from protocol 1)
  • 5% sucrose buffer (see recipe)
  • 25% sucrose buffer (see recipe)
  • Amicon ® Ultra centrifugal concentrator (Merck Millipore), 10 K molecular‐weight cut‐off
  • 4.4‐ml thin‐walled tubes (Sorvall)
  • 27‐G needle
  • 10‐ml syringe
  • Gradient Master IP (Biocomp)
  • TH‐660 (Kendro Laboratory Products GmbH) swing‐out rotors (equivalent to Beckman SW60 rotors)
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Figures

Videos

Literature Cited

Literature Cited
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