The N-terminal end truncated mu-opioid receptor: from expression to circular dichroism analysis.
In order to evaluate the biochemical, biophysical, and pharmacological implication of the N-terminal domain of the human mu-opioid receptor (HuMOR), deletion mutants lacking 64 amino acids from the amino terminus of HuMOR were constructed and expressed in the yeast Pichia pastoris. The recombinant proteins differed with respect to the presence of the Saccharomyces cerevisiae alpha-factor prepropeptide and the enhanced green fluorescent protein fused to the N terminus of the receptor. Pharmacological studies indicated that deletion of the N-terminal domain produced little effect on ligand affinities. The N-terminal end truncated and c-myc/6his-tagged receptor was subsequently purified to homogeneity and a yield of 5 mg/l was obtained after purification. The N-terminal end truncated receptor was further characterized by circular dichroism in trifluoroethanol and showed a characteristic pattern of alpha-helical structure. A pH effect on the structure of the receptor was observed when it was solubilized in sodium dodecyl sulfate micelles, with an increase of helicity at low pH.
Muller
Isabelle
I
IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS, 205 Route de Narbonne, 31077 Toulouse, Cedex 4, France.
Sarramégna
Valérie
V
Milon
Alain
A
Talmont
Franck Jean
FJ
D002942:Circular Dichroism; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017450:Receptors, Opioid, mu / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion / Q000235:genetics*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012995:Solubility; D014270:Trifluoroethanol / Q000737:chemistry
D008823:Micelles; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol