Autoinduction of Protein Expression

Brian G. Fox1, Paul G. Blommel1

1 Department of Biochemistry, Biophysics Degree Program, and Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, Madison, Wisconsin
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.23
DOI:  10.1002/0471140864.ps0523s56
Online Posting Date:  April, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit contains protocols for the use of lactose‐derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X‐ray structural studies, and multi‐well plate growth for screening and optimization are also included. Curr. Protoc. Protein Sci. 56:5.23.1‐5.23.18. © 2009 by John Wiley & Sons, Inc.

Keywords: recombinant protein; expression; Escherichia coli; high‐throughput methods; X‐ray crystallography

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

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Autoinduction of Unlabeled Protein Expression
  • Alternate Protocol 1: Selenomethionine Labeling by Autoinduction
  • Alternate Protocol 2: Expression Screening in an Autoinduction Medium Array
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Autoinduction of Unlabeled Protein Expression

  Materials
  • Sequence‐verified expression plasmid
  • Expression host
  • Agar plates of non‐inducing medium (see recipe)
  • Non‐inducing medium (see recipe)
  • Autoinduction medium (see recipe)
  • Cell suspension buffer (see recipe)
  • 37°C incubator (e.g., New Brunswick C24KC refrigerated shaker or equivalent)
  • 16‐ml snap‐top growth tubes used for culture scale‐up (BD Biosciences) or equivalent
  • 500‐ml Erlenmeyer flasks
  • 25°C shaking incubator
  • 2‐liter polyethyleneterepthalate beverage bottles used for bacterial cell growth (Ball Corporation; standard 2‐liter Erlenmeyer or Fernbock flasks can be substituted for the polyethyleneterepthalate beverage bottles)
  • Allegra 6R centrifuge with a GH3.8 rotor (Beckman Coulter) or equivalent
  • 50‐ml centrifuge tubes

Alternate Protocol 1: Selenomethionine Labeling by Autoinduction

  • Autoinduction medium (lacking vitamin B12 solution and methionine, supplemented with selenomethionine; see recipe)

Alternate Protocol 2: Expression Screening in an Autoinduction Medium Array

  • Sugar‐free methionine‐containing autoinduction medium (see recipe and Table 5.23.1)
  • Sugar‐free selenomethionine‐containing autoinduction medium (see recipe and Table 5.23.1)
  • 96‐well, 2.4 ml per well growth block (Eppendorf)
  • AeraSeal gas‐permeable sealing tape (ISC Bioexpress, T‐2421‐50)
  • Thermomixer R (Eppendorf)
  • 50‐ml Falcon tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bailey, L.J., Elsen, N.L., Pierce, B.S., and Fox, B.G. 2007. Soluble expression and purification of the oxidoreductase component of toluene 4‐monooxygenase. Protein Expr. Purif. 57:9‐16.
   Blommel, P.G. and Fox, B.G. 2005. Fluorescence anisotropy assay for proteolysis of specifically labeled fusion proteins. Anal. Biochem. 336:75‐86.
   Blommel, P.G. and Fox, B.G. 2007. A combined approach to improving large‐scale production of tobacco etch virus protease. Protein Expr. Purif. 55:53‐68.
   Blommel, P.G., Becker, K.J., Duvnjak, P., and Fox, B.G. 2007. Enhanced bacterial protein expression during auto‐induction obtained by alteration of lac repressor dosage and medium composition. Biotechnol. Prog. 23:585‐598.
   Broadwater, J.A. and Fox, B.G. 1998. Spinach acyl carrier protein: Phosphopantetheinylation in Escherichia coli BL21(DE3), in vitro acylation, and enzymatic desaturation of histidine‐tagged isoform I. Protein Expr. Purif. 15:314‐326.
   Dubendorff, J.W. and Studier, F.W. 1991. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219:45‐59.
   Fang, L., Jia, K.Z., Tang, Y.L., Ma, D.Y., Yu, M., and Hua, Z.C. 2007. An improved strategy for high‐level production of TEV protease in Escherichia coli and its purification and characterization. Protein Expr. Purif. 51:102‐109.
   Frederick, R.O., Bergeman, L., Blommel, P.G., Bailey, L.J., McCoy, J.G., Song, J., Meske, L., Bingman, C.A., Riters, M., Dillon, N.A., Kunert, J., Yoon, J.W., Lim, A., Cassidy, M., Bunge, J., Aceti, D.J., Primm, J.G., Markley, J.L., Phillips Jr, G.N., and Fox, B.G. 2007. Small‐scale, semi‐automated purification of eukaryotic proteins for structure determination. J. Struct. Funct. Genomics 8:153‐156.
   Galvao‐Botton, L.M., Katsuyama, A.M., Guzzo, C.R., Almeida, F.C., Farah, C.S., and Valente, A.P. 2003. High‐throughput screening of structural proteomics targets using NMR. FEBS Lett. 552:207‐213.
   Grossman, T.W., Kawasaki, E.S., Punreddy, S.R., and Osborn, M.S. 1998. Spontaneous cAMP‐dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene 209:95‐103.
   Haas, J.A., Frederick, M.A., and Fox, B.G. 2000. Chemical and posttranslational modification of Escherichia coli acyl carrier protein for preparation of dansyl‐acyl carrier proteins. Protein Expr. Purif. 20:274‐284.
   Hoffman, B.J., Broadwater, J.A., Johnson, P., Harper, J., Fox, B.G., and Kenealy, W.R. 1995. Lactose fed‐batch overexpression of recombinant metalloproteins in Escherichia coli BL21(DE3): Process control yielding high levels of metal incorporated, soluble protein. Protein Expr. Purif. 6:646‐654.
   Kennedy, M.A., Montelione, G.T., Arrowsmith, C.H., and Markley, J.L. 2002. Role for NMR in structural genomics. J. Struct. Funct. Genomics 2:155‐169.
   Kumar, R.A., and Clark, D.S. 2006. High‐throughput screening of biocatalytic activity: Applications in drug discovery. Curr. Opin. Chem. Biol. 10:162‐168.
   Levin, E.J., Elsen, N.L., Seder, K.D., McCoy, J.G., Fox, B.G., and Phillips, G.N., Jr. 2008. X‐ray crystal structure of a soluble Rieske‐type ferredoxin from Mus musculus. Acta. Crystallogr. D Biol. Crystallogr. 64:933‐940.
   Lion, N., Reymond, F., Girault, H.H., and Rossier, J.S. 2004. Why the move to microfluidics for protein analysis? Curr. Opin. Biotechnol. 15:31‐37.
   Monod, J. 1972. Nobel Lectures, Physiology or Medicine 1963‐1970. Elsevier Publishing Company, Amsterdam.
   Myers, R.H. and Montgomery, D.C. 2002. Response Surface Methodology: Process and Product Optimization Using Designed Experiments. John Wiley & Sons, New York.
   Neubauer, P., Wolff, C., Hecker, M., Hoffmann, K., Meyer, L., Kruschke, P., and Heinrich, H.W. 1991. Introduction of the tac‐promoter by lactose under fermentation conditions. Acta. Biotechnol. 11:23‐29.
   Neubauer, P., Hofmann, K., Holst, O., Mattiasson, B., and Kruschke, P. 1992. Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as an inducer. Appl. Microbiol. Biotechnol. 36:739‐744.
   Segelke, B. 2005. Macromolecular crystallization with microfluidic free‐interface diffusion. Expert Rev. Proteomics 2:165‐172.
   Sharp, P.M. and Li, W.‐H. 1987. The codon adaptation index ‐ a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 15:1281‐1295.
   Sobrado, P., Goren, M.A., James, D., and Amundson, C.K. 2007. A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles. Protein Expr. Purif. 58:229‐241.
   Sreenath, H.K., Bingman, C.A., Buchan, B.W., Seder, K.D., Burns, B.T., Geetha, H.V., Jeon, W.B., Vojtik, F.C., Aceti, D.J., Frederick, R.O., Phillips, G.N., Jr., and Fox, B.G. 2005. Protocols for production of selenomethionine‐labeled proteins in 2‐L polyethylene terephthalate bottles using auto‐induction medium. Protein Expr. Purif. 40:256‐267.
   Studier, F.W. 2005. Protein production by auto‐induction in high‐density shaking cultures. Protein Expr. Purif. 41:207‐234.
   Studts, J.M. and Fox, B.G. 1999. Application of fed‐batch fermentation to the preparation of isotopically labeled‐ or selenomethionine‐labeled proteins. Protein Expr. Purif. 16:109‐119.
   Studts, J.M., Mitchell, K.H., Pikus, J.D., McClay, K., Steffan, R.J., and Fox, B.G. 2000. Optimized expression and purification of toluene 4‐monooxygenase hydroxylase. Protein Expr. Purif. 20:58‐65.
   Terpe, K. 2006. Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 72:211‐222.
   Thao, S., Zhao, Q., Kimball, T., Steffan, E., Newman, C.S., Fox, B.G., and Wrobel, R.L. 2004. Results from high‐throughput DNA cloning of Arabidopsis thaliana target genes by site‐specific recombination. J. Struct. Funct. Genomics 5:267‐276.
   Tyler, R.C., Sreenath, H.K., Singh, S., Aceti, D.J., Bingman, C.A., Markley, J.L., and Fox, B.G. 2005. Auto‐induction medium for the production of [U‐15N]‐ and [U‐13C, U‐15N]‐labeled proteins for NMR screening and structure determination. Protein Expr. Purif. 40:268‐278.
   van den Berg, S., Lofdahl, P.A., Hard, T., and Berglund, H. 2006. Improved solubility of TEV protease by directed evolution. J. Biotechnol. 121:291‐298.
   van der Woerd, M., Ferree, D., and Pusey, M. 2003. The promise of macromolecular crystallization in microfluidic chips. J. Struct. Biol. 142:180‐187.
   Zheng, B., Gerdts, C.J., and Ismagilov, R.F. 2005. Using nanoliter plugs in microfluidics to facilitate and understand protein crystallization. Curr. Opin. Struct. Biol. 15:548‐555.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library