Purification and characterization of the recombinant extracellular domain of human nerve growth factor receptor expressed in a baculovirus system.
To obtain the large quantities of the extracellular domain of the nerve growth factor receptor (NGF-R) necessary for structural analyses, we produced this protein in the baculovirus expression system. A cDNA coding for the extracellular domain of the human NGF-R was first introduced into transfer vector pVL941. Recombinant baculovirus was produced by cotransfecting Spodoptera frugiperda cells with the transfer vector and DNA of Autographa californica nuclear polyhedrosis virus. Recombinant viral plaques were selected by morphology and dot hybridization. The expression of recombinant extracellular domain (RED) was analyzed by Western blot analysis using anti-NGF-R monoclonal antibody. Insect cells infected with recombinant virus synthesized RED and secreted it into the culture supernatant. RED was isolated by ammonium sulfate precipitation, immunoaffinity chromatography, and anion exchange chromatography yielding 4 mg of RED/liter of suspension culture. Since there was no apparent effect of endoglycosidase H or F on RED electrophoretic mobility, and since RED did not bind concanavalin A or soybean lectin, there appears to be little or no glycosylation of RED. Purified RED bound 125I-NGF as well as anti-NGF-R monoclonal antibodies. Sedimentation analysis and gel exclusion chromatography revealed that RED is an asymmetric molecule and may be a dimer.
Vissavajjhala
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Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Ross
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D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007304:Insect Viruses / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000378:metabolism / Q000382:microbiology; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D014162:Transfection
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004220:Disulfides; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins