Abstract
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.
Highlights
To obtain the large quantities of the extracellular domain of the nerve growth factor receptor (NGF-R)
Secretion of recombinant extracellular domain (RED) into the Culture Supernutant-Sf9 cells infected with AC Hl-3 produced a M, = 40,000 protein which was detected by Western blot analysis with anti-Nerve growth factor (NGF)-R monoclonal antibodies (mAbs) (Fig. 2) but not with a control mAb
RED was prepared in large quantities and compared with native NGF-R
Summary
The anti-NGF-R mAbs bind to conformation-dependent epitopes since they react in Western blotting with nonreduced samples but not reduced samples In these studies, proteins were electrotransferred to Immobilon membranes. Transfer vector pVL Hl-3 coding for RED of human NGF-R (Fig. 1) was constructed as follows. Expression vector pURHl-3 was derived from partial NGF-R cDNA pHl-3 [8] by partial digestion with EcoRI and addition of Hind111 linkers at the 5’ end of the cDNA insert. RED (4 pg/ml) was incubated for 45 min at 37 “C with I*?-NGF (1.0 nM) in Dulbecco’s PBS supplemented with 0.1% bovine serum albumin. A 125~~1 sample including 40 pg of RED, 31 rg of IgG, 25 pg of transferrin, 30 fig of bovine serum albumin, and 25 rg of bovine cytochrome c was layered onto a 5-ml 5-20% (w/v) sucrose density gradient prepared in PBS. Aliquots (0.25 ml) were analyzed by SDS-PAGE and silver staining
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
