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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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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Basic Protocol 1: Large‐Scale Transient Transfection

  • 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

  • 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

  • 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)
  • 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

  • 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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Literature Cited

Literature Cited
  Boussif, O., Lezoualc'h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., & Behr, J. P. (1995). A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America, 92, 7297–7301. doi: 10.1073/pnas.92.16.7297.
  Durocher, Y., & Butler, M. (2009). Expression systems for therapeutic glycoprotein production. Current Opinion in Biotechnology, 20, 700–707. doi: 10.1016/j.copbio.2009.10.008.
  Einhauer, A., & Jungbauer, A. (2001). The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. Journal of Biochemical and Biophysical Methods, 49, 455–465. doi: 10.1016/S0165‐022X(01)00213‐5.
  Gallagher, S. R. (2012). One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science, 68, 10.1.1–10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Gecchele, E., Merlin, M., Brozzetti, A., Falorni, A., Pezzotti, M., & Avesani, L. (2015). A comparative analysis of recombinant protein expression in different biofactories: Bacteria, insect cells and plant systems. Journal of Visualized Experiments, e52459–e52459. doi: 10.3791/52459.
  Geisse, S., & Henke, M. (2005). Large‐scale transient transfection of mammalian cells: A newly emerging attractive option for recombinant protein production. Journal of Structural and Functional Genomics, 6, 165–170. doi: 10.1007/s10969‐005‐2826‐4.
  Ghaderi, D., Zhang, M., Hurtado‐Ziola, N., & Varki, A. (2012). Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non‐human sialylation. Biotechnology and Genetic Engineering Reviews, 28, 147–176. doi: 10.5661/bger‐28‐147.
  Graham, F. L., Smiley, J., Russell, W. C., & Nairn, R. (1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. The Journal of General Virology, 36, 59–74. doi: 10.1099/0022‐1317‐36‐1‐59.
  Griffin, T. J., Seth, G., Xie, H., Bandhakavi, S., & Hu, W‐S. (2007). Advancing mammalian cell culture engineering using genome‐scale technologies. Trends Biotechnol, 25, 401–408. doi: 10.1016/j.tibtech.2007.07.004.
  Hartley, J. L. (2012). Why proteins in mammalian cells? Methods in Molecular Biology, 801, 1–12.. doi: 10.1007/978‐1‐61779‐352‐3_1.
  Hirose, S., Kawamura, Y., Yokota, K., Kuroita, T., Natsume, T., Komiya, K., … Noguchi, T. (2011). Statistical analysis of features associated with protein expression/solubility in an in vivo Escherichia coli expression system and a wheat germ cell‐free expression system. Journal of Biochemistry, 150, 73–81. doi: 10.1093/jb/mvr042.
  Huh, S‐H., Do, H‐J., Lim, H‐Y., Kim, D‐K., Choi, S‐J., Song, H., … Kim, J‐H. (2007). Optimization of 25 kDa linear polyethylenimine for efficient gene delivery. Biologicals, 35, 165–171. doi: 10.1016/j.biologicals.2006.08.004.
  Hutt, D. M., Powers, E. T., & Balch, W. E. (2009). The proteostasis boundary in misfolding diseases of membrane traffic. FEBS Letters, 583, 2639–2646. doi: 10.1016/j.febslet.2009.07.014.
  Itoh, T., Fairall, L., Muskett, F. W., Milano, C. P., Watson, P. J., Arnaudo, N., … Schwabe, J. W. R. (2015). Structural and functional characterization of a cell cycle associated HDAC1/2 complex reveals the structural basis for complex assembly and nucleosome targeting. Nucleic Acids Research, 43, 2033–2044. doi: 10.1093/nar/gkv068.
  Jarvis, D. L. (2009). Baculovirus–Insect Cell Expression Systems. In Guide to Protein Purification (2nd edn, pp. 191–222). Elsevier.
  Lin, Y‐C., Boone, M., Meuris, L., Lemmens, I., Van Roy, N., Soete, A., … Callewaert, N. (2014). Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nature Communications, 5, 4767. doi: 10.1038/ncomms5767.
  Mattanovich, D., Branduardi, P., Dato, L., Gasser, B., Sauer, M., & Porro, D. (2012). Recombinant protein production in yeasts. Methods in Molecular Biology, 824, 329–358.. doi: 10.1007/978‐1‐61779‐433‐9_17.
  Millard, C. J., Watson, P. J., Celardo, I., Gordiyenko, Y., Cowley, S. M., Robinson, C. V., … Schwabe, J. W. R. (2013). Class I HDACs share a common mechanism of regulation by inositol phosphates. Molecular and Cellular, 51, 57–67. doi: 10.1016/j.molcel.2013.05.020.
  Nettleship, J. E., Watson, P. J., Rahman‐Huq, N., Fairall, L., Posner, M. G., Upadhyay, A., … Owens, R. J. (2014). Transient Expression in HEK 293 Cells: An Alternative to E. coli for the Production of Secreted and Intracellular Mammalian Proteins. In E. García‐Fruitós (Ed.) Insoluble proteins (pp. 209–222). New York, NY: Springer New York.
  Portolano, N., Watson, P. J., Fairall, L., Millard, C. J., Milano, C. P., Song, Y., … Schwabe, J. W. R. (2014). Recombinant protein expression for structural biology in HEK 293F suspension cells: A novel and accessible approach. Journal of Visualized Experiments, e51897–e51897. doi: 10.3791/51897.
  Reeves, P. J., Callewaert, N., Contreras, R., & Khorana, H. G. (2002). Structure and function in rhodopsin: High‐level expression of rhodopsin with restricted and homogeneous N‐glycosylation by a tetracycline‐inducible N‐acetylglucosaminyltransferase I‐negative HEK293S stable mammalian cell line. Proceedings of the National Academy of Sciences of the United States of America, 99, 13419–13424. doi: 10.1073/pnas.212519299.
  Rio, D. C., Clark, S. G., & Tjian, R. (1985). A mammalian host‐vector system that regulates expression and amplification of transfected genes by temperature induction. Science, 227, 23–28. doi: 10.1126/science.2981116.
  Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology, 5, 172. doi: 10.3389/fmicb.2014.00172.
  Shaw, G. (2002). Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. The FASEB Journal, 1–20
  Stillman, B. W., & Gluzman, Y. (1985). Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Molecular and Cellular Biology, 5, 2051–2060. doi: 10.1128/MCB.5.8.2051.
  Thomas, P., & Smart, T. G. (2005). HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51, 187–200. doi: 10.1016/j.vascn.2004.08.014.
  Treisman, R. (1985). Transient accumulation of c‐fos RNA following serum stimulation requires a conserved 5‘ element and c‐fos 3’ sequences. Cell, 42, 889–902. doi: 10.1016/0092‐8674(85)90285‐5.
  Varghese, A., Tenbroek, E. M., Coles, J., & Sigg, D. C. (2006). Endogenous channels in HEK cells and potential roles in HCN ionic current measurements. Progress in Biophysics and Molecular Biology, 90, 26–37. doi: 10.1016/j.pbiomolbio.2005.05.002.
  Verma, R., Boleti, E., & George, A. J. (1998). Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems. Journal of Immunological Methods, 216, 165–181. doi: 10.1016/S0022‐1759(98)00077‐5.
  Vink, T., Oudshoorn‐Dickmann, M., Roza, M., Reitsma, J‐J., & de Jong, R. N. (2014). A simple, robust and highly efficient transient expression system for producing antibodies. Methods, 65, 5–10. doi: 10.1016/j.ymeth.2013.07.018.
  Watson, P. J., Fairall, L., & Schwabe, J. W. R. (2012). Nuclear hormone receptor co‐repressors: Structure and function. Molecular and Cellular Endocrinology, 348, 440–449. doi: 10.1016/j.mce.2011.08.033.
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