Two-step purification of full-length nerve growth factor receptor and maintenance of receptor-specific binding activity.
A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification.
Decker
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Upjohn Company, Kalamazoo, Michigan 49001.
Buxser
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D000818:Animals; D002460:Cell Line; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003902:Detergents; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000378:metabolism*; D010713:Phosphatidylcholines; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor; D012995:Solubility
D003902:Detergents; D009414:Nerve Growth Factors; D010713:Phosphatidylcholines; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor