title: Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli.
abstract: Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100. Affinity chromatography using an amylose column and Ni2+ affinity column was used to purify the sigma-1 receptor. The sigma-1 receptor purified by this method is a 26 kDa polypeptide as assessed by SDS-PAGE, binds sigma ligands with high affinity and can be specifically photoaffinity labeled with the sigma-1 receptor photoprobe, [125I]-iodoazidococaine. Ligand binding using [3H]-(+)-pentazocine indicated that approximately half of the purified protein in Triton X-100 bound to radioligand. The MBP-sigma-1 receptor and the sigma-1 receptor in 0.5% triton were maximally stable for approximately two weeks at -20 degrees C in buffer containing 30% glycerol.
authors.affiliation: Department of Pharmacology, University of Wisconsin Medical School, Madison, WI 53706, USA.
authors.forename: Subramaniam
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Hongliang
authors.initials: H
authors.lastname: Lu
authors.forename: Usha
authors.initials: U
authors.lastname: Prabhu
authors.forename: Arnold E
authors.initials: AE
authors.lastname: Ruoho
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D004355:Drug Stability; D004926:Escherichia coli / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006168:Guinea Pigs; D008024:Ligands; D058619:Maltose-Binding Proteins; D010423:Pentazocine / Q000378:metabolism; D017480:Receptors, sigma / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D000097605:Sigma-1 Receptor
chemical_list: D002352:Carrier Proteins; D015951:Factor Xa; D008024:Ligands; D058619:Maltose-Binding Proteins; D010423:Pentazocine; D017480:Receptors, sigma; D011993:Recombinant Fusion Proteins; D000097605:Sigma-1 Receptor