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Purification and recognition of recombinant mouse P2X 1 receptors expressed in a baculovirus system
Author(s) -
Chen Liping,
Hardwick James P.,
McPhie Peter,
Sitkovsky Michail V.,
Jacobson Kenneth A.
Publication year - 2000
Publication title -
drug development research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.582
H-Index - 60
eISSN - 1098-2299
pISSN - 0272-4391
DOI - 10.1002/1098-2299(20000901)51:1<7::aid-ddr2>3.0.co;2-w
Subject(s) - recombinant dna , receptor , biology , virology , microbiology and biotechnology , chemistry , biochemistry , gene
The hexahistidine‐tagged mouse P2X 1 receptor (H‐mP2X 1 R), an ATP‐gated ion channel receptor, was expressed in a baculovirus system using the pAcHLT‐B transfer vector containing a hexahistidine tag. Both widely used denaturing (8M urea) and nondenaturing (such as 1% Triton X‐100) solubilization conditions were compared, resulting in about 30% of the P2X 1 receptors being solubilized (S1). However, at pH 13 most of the H‐mP2X 1 R from the initially insoluble pellet fraction was solubilized (S2) and remained in the soluble fraction (S3) after dialyzing against a nondenaturing buffer. H‐mP2X 1 Rs were purified sequentially through cobalt and ATP affinity columns. Receptors purified from S3 had higher purity than those from S1 (i.e., ∼90% vs. ∼75%). Circular dichroism spectra indicated identical protein secondary structures of the receptors from both sources. Autoradiographic data showed that the purified receptors from S3 had higher affinity for 8‐azido‐ATP‐γ‐ 32 P than the receptors from S1. The binding of 8‐azido‐ATP‐γ‐ 32 P to H‐mP2X 1 R was inhibited by ATP‐γ‐S, α,β‐me‐ATP, and PPADS, but not by a nucleoside analog (N 6 ‐methyl‐2′‐deoxy‐adenosine). In the presence of 2 mM Ca 2+ or Mg 2+ the binding was increased, but not when using a partially purified receptor fraction, in which unidentified proteins bound 8‐azido‐ATP‐γ‐ 32 P or were phosphorylated at 4°C in the presence of 2 mM Mg 2+ . These data suggest that the decrease in potency of ATP in the presence of Ca 2+ and Mg 2+ , as observed in functional studies, is not due to a direct effect of the cations on the binding of ATP to the receptor. Both cyanogen bromide and hydroxylamine cleavage further confirmed the peptide structure of the purified H‐mP2X 1 R. Autoradiographic analysis of the cleavage products showed that 8‐azido‐ATP‐γ‐ 32 P was crosslinked to the carboxyl side of the extracellular domain of the receptor. Drug Dev. Res. 51:7–19, 2000. Published 2000 Wiley‐Liss, Inc.