Wheat Germ Cell‐Free Expression System for Protein Production

Dmitriy A. Vinarov1, Carrie L. Loushin Newman1, Ejan M. Tyler1, John L. Markley1, Mark N. Shahan2

1 University of Wisconsin‐Madison, Madison, Wisconsin, 2 Quintessence Biosciences, Inc., Madison, Wisconsin
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.18
DOI:  10.1002/0471140864.ps0518s44
Online Posting Date:  June, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The Center for Eukaryotic Structural Genomics, in cooperation with Ehime University and CellFree Sciences, has developed a novel wheat germ cell‐free technology for the production of eukaryotic proteins. Protein production and purification are robust and scalable for high‐throughput applications. The protocols have been used to express and purify proteins from Arabidopsis thaliana, human, mouse, rat and zebra fish. This unit describes expression and purification protocols for both small‐scale testing (microgram) and large‐scale production (milligram) of N‐His6‐ and N‐GST‐tagged proteins. The methods described in this unit can be used to produce both unlabeled and labeled proteins required for structure‐based determinations by NMR spectroscopy or X‐ray crystallography.

Keywords: high‐throughput; wheat germ; cell‐free; protein production; NMR structure determination

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Manual Small‐Scale (50‐µl) Transcription Reaction and Ethanol Precipitation of Resultant mRNA
  • Basic Protocol 2: Manual Small‐Scale (50‐µl) Translation Reaction
  • Basic Protocol 3: Manual Large‐Scale (6‐ml) Transcription Reaction and Ethanol Precipitation of Resultant mRNA
  • Basic Protocol 4: Manual Large‐Scale (4‐ml) Translation Reaction and Setup for Automation
  • Basic Protocol 5: Small‐Scale Automated Transcription and Translation Reactions on the GeneDecoder1000
  • Support Protocol 1: Immobilized Metal Affinity Chromatography (IMAC) Purification of His6‐Tagged Fusion Proteins and Preparation of Sample for NMR Analysis
  • Support Protocol 2: Gel‐Filtration Purification and Preparation of Sample for NMR Analysis
  • Support Protocol 3: Purification of GST‐Fusion Protein and Preparation of Sample for NMR Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Manual Small‐Scale (50‐µl) Transcription Reaction and Ethanol Precipitation of Resultant mRNA

  Materials
  • Template DNA construct (purified with Qiagen miniprep kit)
  • 5× transcription buffer (see recipe)
  • 25 mM NTP mixture (see recipe)
  • 80 U/µ RNasin RNase inhibitor solution (Promega)
  • 80 U/µl Sp6 RNA polymerase solution (Promega)
  • Milli‐Q‐purified H 2O
  • 6 M ammonium acetate (sterilize by passing through a 0.2‐µm filter)
  • 70% and 100% ethanol
  • 1.7‐ml microcentrifuge tubes, sterile
  • Benchtop refrigerated centrifuge
  • −80°C freezer

Basic Protocol 2: Manual Small‐Scale (50‐µl) Translation Reaction

  Materials
  • Dialysis buffer with 0.3 mM amino acid mixture, unlabeled (see recipe)
  • mRNA pellet ( protocol 1)
  • Wheat germ cell‐free extract OD 600 200 (CellFree Sciences, http://www.cfsciences.com)
  • 20 mg/ml creatine kinase (Roche) solution in H 2O
  • 80 U/µ RNasin RNase inhibitor solution (Promega)
  • 8 mM amino acid mixture (see recipe)
  • 24‐well plate or dialysis cup holder
  • Dialysis cups (12‐kDa MWCO; Biotech International)
  • Plastic wrap
  • 26°C incubator
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Basic Protocol 3: Manual Large‐Scale (6‐ml) Transcription Reaction and Ethanol Precipitation of Resultant mRNA

  Materials
  • Template DNA construct (purified from plasmid DNA with a Marligen maxiprep kit; http://www.marligen.com)
  • 5× transcription buffer (see recipe)
  • 25 mM NTP mixture
  • 80 U/µ RNasin RNase inhibitor solution (Promega)
  • 80 U/µl Sp6 RNA polymerase solution (Promega)
  • Milli‐Q‐purified H 2O
  • 6 M ammonium acetate (sterile filtered through 0.2 µm filter)
  • 70% and 100% ethanol
  • 50‐ml conical polypropylene centrifuge tube
  • 37°C incubator or water bath
  • Benchtop refrigerated centrifuge
  • −80°C freezer (optional)

Basic Protocol 4: Manual Large‐Scale (4‐ml) Translation Reaction and Setup for Automation

  Materials
  • Dried mRNA pellet ( protocol 3)
  • Wheat germ cell‐free extract OD 600 200 (CellFree Sciences, http://www.cfsciences.com)
  • 16.8 mg/ml creatine kinase (Roche) in 1× dialysis buffer with 0.3 mM amino acids (see recipe)
  • 80 U/µ RNasin RNase inhibitor solution (Promega)
  • Dialysis buffer with 0.3 mM amino acids (see recipe)
  • 15‐ml Amicon centrifugal filtration unit (MWCO 10 kDa; Millipore)
  • Protemist10 (CellFree Sciences, http://www.cfsciences.com)
  • 26°C incubator
  • Benchtop refrigerated centrifuge capable of handling 50‐ml conical tubes
  • 50‐ml conical centrifuge tubes
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)
NOTE: Steps for preparation of the RNA are for both the manual synthesis reaction in a benchtop centrifuge and the automated Protemist10 reaction.

Basic Protocol 5: Small‐Scale Automated Transcription and Translation Reactions on the GeneDecoder1000

  Materials
  • ∼5 µg/ml template DNA constructs (purified from plasmid DNA with a Qiagen miniprep kit)
  • 5× transcription buffer (see recipe)
  • 80 U/µl RNasin RNase inhibitor solution (Promega)
  • 80 U/µl Sp6 RNA polymerase solution (Promega)
  • 25 mM NTP mixture (see recipe)
  • Milli‐Q‐purified H 2O
  • 6 M ammonium acetate (sterilize by passing through a 0.2‐µm filter)
  • 70% and 100% ethanol
  • Dialysis buffer with 0.3 mM amino acids (see recipe)
  • 8 mM amino acid mixture (see recipe)
  • Wheat germ cell‐free extract OD 600 200 (CellFree Sciences; http://www.cfsciences.com)
  • 20 mg/ml creatine kinase (Roche) in H 2O
  • 96‐well PCR plates with lids (Greiner Bio‐One 652280)
  • GeneDecoder1000 and corresponding sterile troughs (CellFree Sciences, http://www.cfsciences.com)
    • MTP plates with lids (Greiner Bio‐One 650201)
    • 20‐µl BioRobotix tips (Molecular BioProducts 918‐261; http://www.mbpinc.com)
    • 300‐µl MBP tips (Molecular BioProducts 3771; http://www.mbpinc.com)
  • Ethanol towels (e.g., Wypall L‐40)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)

Support Protocol 1: Immobilized Metal Affinity Chromatography (IMAC) Purification of His6‐Tagged Fusion Proteins and Preparation of Sample for NMR Analysis

  Materials
  • Reaction mixture ( protocol 4)
  • Protease inhibitor cocktail V (Sigma‐Aldrich)
  • Ni elution buffer (see recipe)
  • Ni binding buffer (see recipe)
  • 1‐ml HisTrap HP chelating column (Amersham 17‐5247‐01)
  • Pump: automated purifier (e.g., ÄKTA; GE Healthcare, http://www.gehealthcare.com) or 1‐ml, 3‐ml, or 10‐ml syringes with a 0.7‐mm blunt end needle, or peristaltic pump
  • Amicon Ultra‐4 concentrators (5‐ or 10‐kDa MWCO; Millipore)
  • Refrigerated centrifuge(s) capable of handling 1.5‐ml microcentrifuge tubes and 15‐ and 50‐ml conical tubes
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and NMR analysis (see unit 17.5)

Support Protocol 2: Gel‐Filtration Purification and Preparation of Sample for NMR Analysis

  Materials
  • NMR screening buffer (see recipe)
  • Glycerol
  • Concentrated sample ( protocol 6)
  • NMR screening buffer with 7% D 2O (see recipe)
  • Protease inhibitor cocktail V (Sigma‐Aldrich)
  • Superdex 75 column (GE Healthcare, http://www.gehealthcare.com)
  • Pump: automated purifier (e.g., ÄKTA; GE Healthcare, http://www.gehealthcare.com) or 1‐ml, 3‐ml, or 10‐ml syringes with a 0.7‐mm blunt end needle, or peristaltic pump
  • Refrigerated centrifuge
  • Amicon Ultra‐4 concentrators (5‐ or 10 kDa MWCO)
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), protein quantitation (unit 3.4), and NMR analysis (see unit 17.5)

Support Protocol 3: Purification of GST‐Fusion Protein and Preparation of Sample for NMR Analysis

  Materials
  • Phosphate‐buffered saline (PBS; diluted from 10× PBS; see recipe) with 10 mM dithiothreitol (DTT): PBS+DTT
  • 10× PBS (see recipe)
  • GST‐fusion construct ( protocol 4)
  • 10 mM reduced glutathione (Sigma, G‐6529) in PBS+DTT
  • NMR screening buffer (see recipe)
  • 10× protease buffer, pH 7 (see recipe)
  • PreScission protease solution (GE Healthcare)
  • NMR screening buffer with 7% D 2O (see recipe)
  • BCA protein assay reagents (Pierce)
  • 1‐ml GSTrap HP column (Amersham 17‐5281‐01 or 17‐5282‐02) or GST Microspin purification kit (Amersham, 27‐4570‐03)
  • Pump: automated purifier (e.g., ÄKTA; GE Healthcare, http://www.gehealthcare.com) or 1‐ml, 3‐ml, or 10‐ml syringes with a 0.7‐mm blunt end needle, or peristaltic pump
  • Refrigerated centrifuge
  • Amicon Ultra‐4 concentrator (5 or 10 kDa; Millipore)
  • 1.5‐ml microcentrifuge tubes
  • Nutator mixer (Clay Adams)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), NMR analysis (see unit 17.5), and protein quantitation (unit 3.4)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Betton, J.M. 2003. Rapid translation system (RTS): A promising alternative for recombinant protein production. Curr. Protein Pept. Sci. 4:73‐80.
   Bruggert, M., Rehm, T., Shanker, S., Georgescu, J., and Holak, T.A. 2003. A novel medium for expression of proteins selectively labeled with 15N‐amino acids in Spodoptera frugiperda (Sf9) insect cells. J. Biomol. NMR 25:335‐348.
   Chrunyk, B.A., Evans, J., Lillquist, J., Young, P., and Wetzel, R. 1993. Inclusion‐body formation and protein stability in sequence variants of interleukin‐1‐beta. J. Biol. Chem. 268:18053‐18061.
   Chumpolkulwong, N., Hori‐Takemoto, C., Hosaka, T., Inaoka, T., Kigawa, T., Shirouzu, M., Ochi, K., and Yokoyama, S. 2004. Effects of Escherichia coli ribosomal protein S12 mutations on cell‐free protein synthesis. Eur. J. Biochem. 271:1127‐1134.
   Clemens, M.M. and Prujin, G.J. 1999. In Protein Expression. A practical approach. (S.J. Higgens. and B.D. Hames, eds.) pp. 129‐165. Oxford University Press, Oxford.
   Cubeddu, L., Moss, C.X., Swarbrick, J.D., Gooley, A.A., Williams, K.L., Curmi, P.M., Slade, M.B., and Mabbutt, B.C. 2000. Dictyostelium discoideum as expression host: Isotopic labeling of a recombinant glycoprotein for NMR studies. Protein Expr. Purif. 19:335‐342.
   Goff, S.A. and Goldberg, A.L. 1987. An increased content of protease LA, the lon gene‐product, increases protein‐degradation and blocks growth in Escherichia coli. J. Biol. Chem. 262:4508‐4515.
   Guignard, L., Ozawa, K., Pursglove, S.E., Otting, G., and Dixon, N.E. 2002. NMR analysis of in vitro‐synthesized proteins without purification: A high‐throughput approach. FEBS Lett. 524:159‐162.
   Henrich, B., Lubitz, W., and Plapp, R. 1982. Lysis of Escherichia coli by induction of cloned Phi‐X174 genes. Mol. Gen. Genet. 185:493‐497.
   Kawasaki, T., Gouda, M.D., Sawasaki, T., Takai, K., and Endo, Y. 2003. Efficient synthesis of a disulfide‐containing protein through a batch cell‐free system from wheat germ. Eur. J. Biochem. 270:4780‐4786.
   Kigawa, T. and Yokoyama, S. 2002. High‐throughput cell‐free protein expression system for structural genomics and proteomics studies. Tanpakushitsu Kakusan Koso 47:1014‐1019.
   Kigawa, T., Muto, Y., and Yokoyama, S. 1995. Cell‐free synthesis and amino acid‐selective stable isotope labeling of proteins for NMR analysis. J. Biomol. NMR 6:129‐134.
   Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., and Yokoyama, S. 1999. Cell‐free production and stable‐isotope labeling of milligram quantities of proteins. FEBS Lett. 442:15‐19.
   Kim, D.M. and Swartz, J.R. 2000. Prolonging cell‐free protein synthesis by selective reagent additions. Biotechnol. Prog. 16:385‐390.
   Kim, D.M., Kigawa, T., Choi, C.Y., and Yokoyama, S. 1996. A highly efficient cell‐free protein synthesis system from Escherichia coli. Eur. J. Biochem. 239:881‐886.
   Klammt, C., Lohr, F., Schafer, B., Haase, W., Dotsch, V., Ruterjans, H., Glaubitz, C., and Bernhard, F. 2004. High level cell‐free expression and specific labeling of integral membrane proteins. Eur. J. Biochem. 271:568‐580.
   Kramer, G., Kudlicki, W., and Hardesty, B. 1999. Cell‐free coupled transcription‐translation systems from Escherichia coli. In Protein Expression: A Practical Approach (S.J. Higgens and B.D. Hames, eds.) pp. 201‐223. Oxford University Press, Oxford.
   Madin, K., Sawasaki, T., Ogasawara, T., and Endo, Y. 2000. A highly efficient and robust cell‐free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Sci. U.S.A. 97:559‐564.
   Maurizi, M.R. 1987. Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli. J. Biol. Chem. 262:2696‐2703.
   Morita, E.H., Sawasaki, T., Tanaka, R., Endo, Y., and Kohno, T. 2003. A wheat germ cell‐free system is a novel way to screen protein folding and function. Protein Sci. 12:1216‐1221.
   Sawasaki, T., Hasegawa, Y., Tsuchimochi, M., Kamura, N., Ogasawara, T., Kuroita, T., and Endo, Y. 2002a. A bilayer cell‐free protein synthesis system for high‐throughput screening of gene products. FEBS Lett. 514:102‐105.
   Sawasaki, T., Ogasawara, T., Morishita, R., and Endo, Y. 2002b. A cell‐free protein synthesis system for high‐throughput proteomics. Proc. Natl. Acad. Sci. U.S.A. 99:14652‐14657.
   Shi, J., Pelton, J.G., Cho, H.S., and Wemmer, D.E. 2004. Protein signal assignments using specific labeling and cell‐free synthesis. J. Biomol. NMR 28:235‐247.
   Strauss, A., Bitsch, F., Cutting, B., Fendrich, G., Graff, P., Liebetanz, J., Zurini, M., and Jahnke, W. 2003. Amino‐acid‐type selective isotope labeling of proteins expressed in Baculovirus‐infected insect cells useful for NMR studies. J. Biomol. NMR 26:367‐372.
   Torizawa, T., Terauchi, T., and Kainosho, M. 2002. Recent developments in NMR methods for structural biology. Seikagaku 74:1279‐1284.
   Tyler, R.C., Aceti, D.J., Bingman, C.A., Cornilescu, C.C., Fox, B.G., Frederick, R.O., Jeon, W.B., Lee, M.S., Newman, C.S., Peterson, F.C., Phillips, G.N., Shahan, M.N., Singh, S., Song, J., Sreeenath, H.K., Tyler, E.M., Ulrich, E.L., Vinarov, D.A., Vojtik, F.C., Volkman, B.F., Wrobel, R.L., Zhao, Q., and Markley, J.L. 2005. Comparison of cell‐based and cell‐free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies. Proteins 59:633‐643.
   Vinarov, D.A., Lytle, L.B., Peterson, F.C., Tyler, E.M., Volkman, B.F., and Markley, J.L. 2004. Eukaryotic cell‐free structural genomics: NMR structure of a beta‐grasp fold protein from Arabidopsis thaliana. Nat. Methods 1:149‐153.
   Yabuki, T., Kigawa, T., Dohmae, N., Takio, K., Terada, T., Ito, Y., Laue, E.D., Cooper, J.A., Kainosho, M., and Yokoyama, S. 1998. Dual amino acid‐selective and site‐directed stable‐isotope labeling of the human c‐Ha‐Ras protein by cell‐free synthesis. J. Biomol. NMR 11:295‐306.
   Yin, G. and Swartz, J.R. 2004. Enhancing multiple disulfide bonded protein folding in a cell‐free system. Biotechnol. Bioeng. 86:188‐195.
   Yokoyama, S. 2003. Protein expression systems for structural genomics and proteomics. Curr. Opin. Chem Biol. 7:39‐43.
   Yokoyama, S., Hirota, H., Kigawa, T., Yabuki, T., Shirouzu, M., Terada, T., Ito, Y., Matsuo, Y., Kuroda, Y., Nishimura, Y., Kyogoku, Y., Miki, K., Masui, R., and Kuramitsu, S. 2000. Structural genomics projects in Japan. Nat. Struct. Biol. 7:943‐945.
Key References
   Kigawa et al., 1995. See above.
  Discusses the applicability and advantages of E. coli cell‐free protein synthesis for selective stable isotope labeling of proteins for the application of multidimensional NMR spectroscopy to larger proteins.
   Klammt et al., 2004. See above.
  The authors demonstrate high level expression of integral membrane proteins (IMPs) in a cell‐free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation.
   Madin et al., 2000. See above.
  One of the original papers by Endo and colleagues describing the development and evolution of the wheat germ cell‐free protein production.
   Tyler et al., 2005. See above.
  The authors describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell‐based and wheat germ cell‐free protocols.
   Vinarov et al., 2004. See above.
  Describes a wheat germ cell‐free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell‐based methods.
   Yokoyama, 2003. See above.
  Discusses application of the E. coli cell‐free protein synthesis system for large‐scale production of protein samples for NMR (stable‐isotope labeling) and X‐ray crystallography (selenomethionine substitution).
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library