Purification and characterization of the P-80 glycoprotein from human brain.
Ontology highlight
ABSTRACT:
SUBMITTER: Girgrah N
PROVIDER: S-EPMC1135416 | biostudies-other | 1988 Dec
REPOSITORIES: biostudies-other
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Project description:Human P-glycoprotein (P-gp) is an ATP-binding cassette transporter that has been implicated in altering the pharmacokinetics of anticancer drugs in normal tissues and development of multidrug resistance in tumor cells via drug efflux. There is still no definitive explanation of the mechanism by which P-gp effluxes drugs. One of the challenges of large-scale purification of membrane transporters is the selection of a suitable detergent for its optimal extraction from cell membranes. In addition, further steps of purification can often lead to inactivation and aggregation, decreasing the yield of purified protein. Here we report the large-scale purification of human P-gp expressed in High-Five insect cells using recombinant baculovirus. The purification strategies we present yield homogeneous functionally active wild type P-gp and its E556Q/E1201Q mutant, which is defective in carrying out ATP hydrolysis. Three detergents (1,2-diheptanoyol-sn-glycero-3-phosphocholine, dodecyl maltoside and n-octyl-β-d-glucopyranoside) were used to solubilize and purify P-gp from insect cell membranes. P-gp purification was performed first using immobilized metal affinity chromatography, then followed by a second step of either anion exchange chromatography or size exclusion chromatography to yield protein in concentrations of 2-12 mg/mL. Size exclusion chromatography was the preferred method, as it allows separation of monomeric transporters from aggregates. We show that the purified protein, when reconstituted in proteoliposomes and nanodiscs, exhibits both basal and substrate or inhibitor-modulated ATPase activity. This report thus provides a convenient and robust method to obtain large amounts of active homogeneously purified human P-gp that is suitable for biochemical, biophysical and structural characterization.
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Project description:PurposeThe goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones.Materials and methodsFor hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus.ResultsTwelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953-7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection.ConclusionThis study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.
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