Rapid Screening of E. coli Extracts by Heteronuclear NMR

Angela M. Gronenborn1

1 National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.11
DOI:  10.1002/0471140864.ps0711s31
Online Posting Date:  May, 2003
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Abstract

Assessing whether a protein or protein complex is amenable to structural analysis is an important component in the structural genomics effort. In particular, if complete sets of structures for entire genomes are to be obtained within a reasonable time frame, high throughput methodologies for all steps along the way have to be developed. These days, cloning and expression systems are highly optimized and a variety of commercially available vectors can be used. However, heterologous proteins or protein domains expressed in bacteria may not be soluble or correctly folded, necessitating intricate solubilization and refolding schemes prior to structural or functional studies. NMR spectroscopy is an important tool for assessing the solubility, stability, and structural integrity of a gene product. It allows efficient evaluation of many variations of polypeptide length and sequence (without time‐ and labor‐intensive purification/refolding procedures) using 1H‐15N‐HSQC spectroscopy of samples of 15N‐labeled proteins directly from crude E. coli extracts. In addition to screening for particular properties of the expressed protein alone, it is also possible to map intermolecular interactions such as ligand binding. In this unit, the basic methodology for bacterial growth, isotope labeling, and spectroscopic evaluation of the protein structure is provided.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: 15N Protein Labeling and NMR Characterization
  • Alternate Protocol 1: Enrichment of 15N‐Labeled Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: 15N Protein Labeling and NMR Characterization

  Materials
  • Bacteria (E. coli strains BL21(DE3) or HMS174) containing the expression plasmid (GEV1 or GEV2) carrying the coding sequence for the protein of interest
  • recipeMinimal medium (see recipe)
  • 1 M IPTG
  • PBS, pH 7.4
  • 20 mM sodium phosphate, pH 5.4
  • D 2O
  • Shaker incubator (New Brunswick Scientific)
  • French pressure cell (unit 6.7); alternatively the bacteria can be ruptured in a microfluidizer
  • Concentrators (e.g., Centriprep‐3, Amicon)
  • NMR spectrometer

Alternate Protocol 1: Enrichment of 15N‐Labeled Protein

  • 50 mM Tris⋅Cl, pH 7.5/5 mM EDTA/5 mM benzamidine solution
  • 20‐ml IgG Sepharose fast‐flow column
  • 50 mM Tris⋅Cl, pH 7.5 /150 mM NaCl
  • 5 mM sodium acetate buffer, pH 5.0
  • 5 M sodium acetate buffer, pH 3.5
  • 10 to 20 mM sodium phosphate, pH 5 to 6
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
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   Creemers, A.F., Klaassen, C.H., Bovee‐Geurts, P.H., Kelle, R., Kragl, U., Raap, J., de Grip, W.J., Lugtenburg, J., and de Groot, H.J. 1999. Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G‐protein‐coupled receptor rhodopsin. Biochemistry 38:7195‐7199.
   de Lamotte, F., Boze, H., Blanchard, C., Klein, C., Moulin, G., Gautier, M.F., and Delsuc, M.A. 2001. NMR monitoring of accumulation and folding of 15N‐labeled protein overexpressed in Pichia pastoris. Protein Expr. Purif. 22:318‐324.
   Gronenborn, A.M. and Clore, G.M. 1996. Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy. Protein Sci. 5:174‐177.
   Gronenborn, A.M., Frank, M.K., and Clore, G.M. 1996. Core mutants of the immunoglobulin binding domain of Streptococcal protein G: Stability and structural integrity. FEBS Lett. 398:312‐316.
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   Muchmore, D.C., McIntosh, L.P., Russell, C.B., Anderson, D.E., and Dahlquist, F.W. 1989. Expression and nitrogen‐15 labeling of proteins for proton and nitrogen‐15 nuclear magnetic resonance. Methods Enzymol. 177:44‐73.
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   Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorf, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:60‐89.
   Zhou, P., Lugovskoy, A.A., and Wagner, G. 2000. A solubility‐enhancement tag (SET) for NMR studies of poorly behaving proteins. J. Biomol.NMR 20:11‐14.
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