کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2021470 1069247 2007 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli
چکیده انگلیسی

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 °C in buffer containing 30% glycerol.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 51, Issue 2, February 2007, Pages 283–292
نویسندگان
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