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Autoinduction of Protein Expression

Brian G. Fox1,  Paul G. Blommel1

1Department 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
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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

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: 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
     
 
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Materials

Basic Protocol: 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

 Additional Materials (also see the Basic Protocol)
  • Autoinduction medium (lacking vitamin B12 solution and methionine, supplemented with selenomethionine; see recipe)

Alternate Protocol 2: Expression Screening in an Autoinduction Medium Array

 Additional Materials (also see the Basic Protocol)
  • 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
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Figures

  • Figure 5.23.1
    Denaturing electrophoresis gels showing results from autoinduced expression, OD600 values, and elapsed time of the growth. (A) Toluene 4-monooxygenase hydroxylase [210 kDa with ()2 quaternary structure composed of TmoA (55 kDa), TmoB (10 kDa), and TmoE (35 kDa) polypeptides (Studts et al., 2000)]. Vector pVP58K was derived from pQE80 to include the Flexi Vector cloning cassette (Promega) and a kanamycin selectable marker. The OD600 values obtained during the autoinduction in a shaken-flask culture are shown. Enzyme produced in this way had kcat of ~3 sec––1, which is comparable to the best preparations obtained from any induction system. (B) Soluble Rieske-type ferredoxin sMR from mouse (gene Mm266515) expressed in a shaken-flask culture as a fusion to the C-terminus of His8-maltose binding protein [63 kDa, PDB ID 3D89, (Levin et al., 2008)]. Vector pVP16A was derived from pQE80 to include the Gateway cloning cassette (Invitrogen) and an ampicillin selectable marker (Thao et al., 2004). Tight control of basal expression at OD600 of 2 and 3 and high-level expression at OD600 of 33 are demonstrated. The harvested cells were dark brown, corresponding to incorporation of an iron-sulfur center into the expressed ferredoxin. (C) Toluene 4-monooxygenase hydroxylase expressed from pVP58K in a fermenter with autoinduction medium enriched with 57Fe for studies using Mössbauer spectroscopy. The dissolved O2 was fixed at 10% and early onset of expression (between OD600 of 2 and 3) and continued accumulation of active enzyme up to an OD600 of 14 were observed. Approximately 90 g of wet cells were obtained from 10 liters of autoinduction medium. The purified enzyme yield was ~14 mg per gram of cells, or ~130 mg per liter of medium.

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  • Figure 5.23.2
    A picture of a 96-well plate containing diluted lysates obtained from autoinduction of enhanced green fluorescent protein expression using the expression media array defined in Tables 5.23.1 and 5.23.2. The plate was illuminated with a 340-nm light source.

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  • Figure 5.23.3
    Time course of autoinduction of the expression of a fusion of tobacco etch virus (TEV) protease to maltose-binding protein (MBP) in a fermenter. The expressed fusion protein had a protease cleavage site located in the linker region between maltose-binding protein and the protease, which yielded self-cleavage of the fusion protein during the cell growth. The times when samples were obtained and their OD600 values are indicated. The samples at 8.7 hr are the total cell lysate (T) and the soluble fraction (S) obtained from lysed cells; >95% of the total protease was found in the soluble fraction.

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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.
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    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.
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    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.
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    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.
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    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.
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matthew w vetting (not verified)
Posted on May 27, 2009 - 9:25am

Some of the media formulations are not correct.

The autoinduction formula is supposed to have 0.03% glucose, 0.45% lactose, 0.9% glycerol

The listed autoinduction formula has 0.5% glucose, 1% lactose, 1% glycerol

Some of the media says to add 10 mls of 50x amino acids per liter (should either be 100x amino acids or 20 mls of 50x amino acids)

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