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Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
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JoVE Journal Química
Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

DOI:
10.3791/50141-v

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12:05 min

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March 06, 2013

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, , , , ,
1Structural Biology Division,The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne

Capítulos

  • 00:05Título
  • 02:06Inclusion Body Preparation and Nickel Matrix Binding
  • 03:55On-column Oxidative Crosslinking
  • 04:50TFA Elution and Quantitation
  • 05:52CNBr Digestion
  • 07:54Reversed-phase HPLC Purification of Disulfide-crosslinked Peptide Complexes
  • 09:06TrpLE fusions: Transmembrane Peptide Complexes Expressed in Bacteria
  • 11:19Conclusion

Summary

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Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Tags

Keywords: Disulfide-stabilizedTransmembrane Peptide ComplexesStructural StudiesMembrane ProteinsTM DomainsTM SignalingTM Peptide ComplexesHomo-dimericHetero-dimericStable Isotope LabelsNMRNon-natural Amino AcidsDisulfide-crosslinkedDetergentLipidMembrane-mimetic

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