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Titolo:
EXPRESSION AND CHARACTERIZATION OF THE HUMAN ERYTHROCYTE ANION-EXCHANGER IN A BACULOVIRUS SF-9 CELL SYSTEM/
Autore:
DALE WE; TEXTOR JA; MERCER RW; SIMCHOWITZ L;
Indirizzi:
VET AFFAIRS MED CTR,DEPT MED ST LOUIS MO 63110 WASHINGTON UNIV,SCH MED,DEPT MED ST LOUIS MO 63110 WASHINGTON UNIV,SCH MED,DEPT CELL BIOL & PHYSIOL ST LOUIS MO 63110
Titolo Testata:
Protein expression and purification
fascicolo: 1, volume: 7, anno: 1996,
pagine: 1 - 11
SICI:
1046-5928(1996)7:1<1:EACOTH>2.0.ZU;2-M
Fonte:
ISI
Lingua:
ENG
Soggetto:
RED BLOOD-CELLS; BAND-3 PROTEIN; INSECT CELLS; FUNCTIONAL EXPRESSION; TRANSPORT KINETICS; XENOPUS OOCYTES; MEMBRANE; LOCALIZATION; MECHANISM; LAEVIS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
46
Recensione:
Indirizzi per estratti:
Citazione:
W.E. Dale et al., "EXPRESSION AND CHARACTERIZATION OF THE HUMAN ERYTHROCYTE ANION-EXCHANGER IN A BACULOVIRUS SF-9 CELL SYSTEM/", Protein expression and purification, 7(1), 1996, pp. 1-11

Abstract

The human erythrocyte anion-exchange protein (HAE1) has been expressed in insect Sf-9 cells using a recombinant baculovirus. We subcloned the full-length cDNA encoding HAE1 into the baculovirus expression vector pVL1392 and cotransfected Sf-9 cells with the recombinant vector and wild-type AcMNPV DNA to obtain recombinant baculovirus. The expressed protein was targeted to the Sf-9 plasma membrane at an apparent density of similar to 0.5 x 10(6) copies/cell as determined by quantitative autoradiography using an HAE1-specific monoclonal antibody. Unlike native HAE1, the expressed protein was not glycosylated. Transport studies with HAE1-recombinant-infected Sf-9 cells showed saturable [K-m(Cl-) = 44 V-max; (Cl-) = 48 mEq/liter of cell water . min] and H2DIDS-inhibitable (K-0.5 = 34 mu M) Cl-36(-) uptake that was not present in uninfected cells. We also found that extracellular SO42- reduced Cl-36(-) influx [K-0.5(SO42-) = 26 mM], presumably through substrate competition as in erythrocytes. Finally, we observed that H2DIDS-inhibitable Cl-36(-) efflux was reduced by 77% in the nominal absence of a suitablecounter-anion in the external solution (HCO3--free, all-glucuronate medium), thereby providing strong evidence for an obligatory exchange mechanism. We conclude that there is high-level expression of HAE1 functional activity in recombinant baculovirus-infected Sf-9 cells and that this system will prove useful for kinetic and structural analyses ofthe HAE1 protein. (C) 1996 Academic Press, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 20/01/21 alle ore 15:58:22