Synthesis of the C-terminal region of opioid receptor like 1 in an SDS micelle by the native chemical ligation: effect of thiol additive and SDS concentration on ligation efficiency

J Pept Sci. 2005 Jul;11(7):410-6. doi: 10.1002/psc.634.

Abstract

In the process of developing a method for the synthesis of membrane proteins, the conditions for native chemical ligation, namely, detergent concentration and the chemical characteristics of the thiol additive were investigated in detail. The C-terminal region of the opioid receptor like 1, ORL1(288-370), which contains C-terminal intracellular and transmembrane domains, was chosen as a model. The building blocks, ORL1(329-370) and ORL1(288-328)-SR-Gly-Arg(5)-Leu (-SR- : -SCH(2)CH(2)CO-) were most effectively condensed slightly below the critical micelle concentration of SDS and in the presence of mercaptoethanesulfonic acid as a thiol additive. The results showed that the concentration of SDS and the charge on the thiol additive are crucial factors for the effective synthesis of a membrane protein by native chemical ligation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Arginine / metabolism
  • Chromatography, High Pressure Liquid
  • Fluorenes / chemistry
  • Mercaptoethanol / analogs & derivatives*
  • Mercaptoethanol / chemistry*
  • Micelles*
  • Molecular Sequence Data
  • Nociceptin Receptor
  • Peptide Fragments / biosynthesis*
  • Receptors, Opioid / biosynthesis*
  • Receptors, Opioid / chemistry
  • Receptors, Opioid / metabolism
  • Sodium Dodecyl Sulfate*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Sulfhydryl Compounds / chemistry*

Substances

  • Amino Acids
  • Fluorenes
  • Micelles
  • N(alpha)-fluorenylmethyloxycarbonylamino acids
  • Peptide Fragments
  • Receptors, Opioid
  • Sulfhydryl Compounds
  • Sodium Dodecyl Sulfate
  • Mercaptoethanol
  • Arginine
  • Nociceptin Receptor