Detection, Purification, and Characterization of cDNA Clones Encoding DNA‐Binding Proteins

Harinder Singh1

1 University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.7
DOI:  10.1002/0471142727.mb1207s13
Online Posting Date:  May, 2001
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Abstract

In this unit, an appropriate recombinant clone is detected in an expression library with a DNA recognition‐site probe, purified, and shown to encode a DNA‐binding domain of defined sequence specificity. The strategy described below obviates purification of a sequence‐specific DNA‐binding protein for the purpose of isolating its gene; it simply requires a suitable cDNA library constructed in the expression vector lgt11 and a DNA recognition‐site probe. The basic protocol enables the detection and purification of clones encoding sequence‐specific DNA‐binding proteins. The alternate protocol describes a denaturation/renaturation procedure that can increase detection of certain clones. The support protocol provides a rapid means of characterizing the DNA‐binding activities of the proteins encoded by the cloned cDNAs.

     
 
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Table of Contents

  • Basic Protocol 1: Screening a λgt11 Expression Library with Recognition‐Site DNA
  • Alternate Protocol 1: Denaturation/Renaturation Cycling of Dried Replica Filters Using Guanidine⋅HCl
  • Support Protocol 1: Preparation of a Crude Extract from λgt11 Recombinant Lysogen to Characterize DNA‐Binding Activity of the Fusion Protein
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Screening a λgt11 Expression Library with Recognition‐Site DNA

  Materials
  • pUC recombinant plasmid DNA containing multiple tandem copies of recognition‐site DNA and lacking the recognition site (or containing mutant versions; units 1.5 & 3.16; see 12.7commentary)
  • EcoRI and HindIII restriction endonucleases (unit 3.1)
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 22)
  • 5 mM each dCTP, dGTP, dTTP, and dATP (unit 3.4)
  • 5000 Ci/mmol [α‐32P]dATP
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • 500 mM EDTA, pH 8.0
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 24:1 chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol
  • recipeElution buffer
  • E. coli Y1090 (Table 97.80.4711)
  • λgt11 cDNA library (unit 5.8; see commentary)
  • LB medium containing 0.2% maltose and 50 µg/ml ampicillin (unit 1.1 and Table 97.80.4711)
  • 0.7% top agarose, melted and equilibrated to 47°C (unit 1.1)
  • 100‐ and 150‐mm LB plates containing 50 µg/ml ampicillin, dry and prewarmed to 37°C (unit 1.1 and Table 97.80.4711)
  • 10 mM IPTG (prepare 1 M stock with sterile H 2O and store at −20°C; Table 97.80.4711)
  • recipeBLOTTO
  • recipeBinding buffer
  • 1 mg/ml sonicated (to ∼1 Kb) calf thymus DNA
  • Chloroform
  • Elutip‐d columns ( Schleicher & Schuell)
  • 37° and 42°C incubators
  • 132‐ and 82‐mm nitrocellulose membrane filters ( Schleicher & Schuell)
  • Additional reagents and equipment for restriction digest (unit 3.1), mobility shift DNA‐binding assay (unit 12.2), phenol extraction and ethanol precipitation (unit 2.1), nondenaturing polyacrylamide gel electrophoresis (unit 2.7), Elutip‐d chromatography (unit 2.6), titering and plating bacteriophage (units 1.11 & 6.1), 3.NaNautoradiography ( 3.NaN), and purifying and storing bacteriophage clones (units 6.5 & 1.12)

Alternate Protocol 1: Denaturation/Renaturation Cycling of Dried Replica Filters Using Guanidine⋅HCl

  Additional Materials
  • recipeHEPES binding buffers

Support Protocol 1: Preparation of a Crude Extract from λgt11 Recombinant Lysogen to Characterize DNA‐Binding Activity of the Fusion Protein

  Additional Materials
  • E. coli Y1089 hflA150 (Table 97.80.4711)
  • LB medium with and without 10 mM MgCl 2 (unit 1.1)
  • 1010 pfu/ml λgt11 recombinant phage stock (see protocol 1basic protocol)
  • 1 M IPTG (Table 97.80.4711)
  • recipeExtract buffer
  • 5 mg/ml lysozyme in recipeextract buffer (store at −20°C)
  • 4 M NaCl
  • 32°C incubator
  • Sterile toothpicks
  • 0.025‐µm filter disks (Millipore VS)
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Figures

Videos

Literature Cited

Literature Cited
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Key Reference
   Huynh, T.V., Young, R.A., and Davis, R.W. 1985. Construction and screening cDNA libraries in λgt10 and λgt11. In DNA Cloning, Vol. 1: A Practical Approach (D.M. Glover, ed.) pp.49‐78. IRL Press, Oxford.
  Provides an excellent description of the λgt11 expression screening system.
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