Synthesizing Proteins In Vitro by Transcription and Translation of Cloned Genes

Kevin Struhl1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.17
DOI:  10.1002/0471142727.mb1017s48
Online Posting Date:  May, 2001
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Abstract

The availability of a cloned gene makes it possible to synthesize the encoded protein by in vitro transcription and translation. The basis of the method presented in this unit is to clone the protein‐coding sequences into a vector containing a promoter for SP6 or T7 RNA polymerase, to produce messenger RNA by transcribing the DNA template with this enzyme, and to synthesize the desired protein (often as an 35S‐labeled species) by translation of this mRNA in vitro. A major advantage of this method is that any desired mutant protein can be generated simply by altering the DNA template.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Plasmid DNA containing SP6 or T7 promoter
  • DNA containing cloned gene or cDNA encoding protein of interest
  • Appropriate restriction endonucleases (unit 3.1)
  • TE buffer ( appendix 22)
  • recipe5× ribonucleoside triphosphate mix
  • 10× SP6/T7 RNA polymerase buffer (unit 3.4)
  • 10 mM spermidine (for SP6 RNA polymerase only)
  • Pancreatic ribonuclease inhibitor (e.g., RNasin from Promega Biotec)
  • SP6 or T7 RNA polymerase (unit 3.8)
  • Buffered phenol (unit 2.1)
  • Isobutanol
  • 10 M ammonium acetate
  • 100% ethanol
  • In vitro translation kit (wheat germ extract or reticulocyte lysate)
  • 35S‐labeled methionine (1400 Ci/mmol)
  • 0.1 M NaOH
  • 10% trichloroacetic acid (TCA)
  • EN3HANCE ( Du Pont NEN)
  • Additional materials for subcloning DNA fragments (unit 3.16), preparing highly purified plasmid DNA (unit 1.7), digesting DNA with restriction endonucleases (unit 3.1), phenol extraction and ethanol precipitation (unit 2.1), agarose and nondenaturing polyacrylamide gel electrophoresis (units 2.5 2.7 & 12.2), quantitating radioactivity by acid precipitation on glass‐fiber filters (unit 3.4), one‐dimensional SDS‐PAGE (unit 10.2), and autoradiography ( 3.NaN).
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Figures

Videos

Literature Cited

Literature Cited
   Hope, I.A. and Struhl, K. 1985. GCN4 protein synthesized in vitro, binds HIS3 regulatory sequences: Implications for general control of amino acid biosyntheic genes in yeast. Cell 43:177‐188.
   Hope, I.A. and Struhl, K. 1986. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell 46:885‐894.
   Hope, I.A. and Struhl, K. 1987. GCN4, a eukaryotic transcriptional activator protein, binds DNA as a dimer. EMBO J. 6:2781‐2784.
   Hope, I.A., Mahadevan, S., and Struhl, K. 1988. Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein. Nature 333:635‐640.
   Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12:7057‐7070.
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