Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.
For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.
Kim
Tae-Kang
TK
Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40292, USA.
Zhang
Rundong
R
Feng
Wenke
W
Cai
Jian
J
Pierce
William
W
Song
Zhao-Hui
ZH
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008024:Ligands; D008565:Membrane Proteins; D043884:Receptor, Cannabinoid, CB1; D011993:Recombinant Fusion Proteins; D000432:Methanol