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
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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 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
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Basic Protocol 1: Autoinduction of Unlabeled Protein Expression

  • 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
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Literature Cited

Literature Cited
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