Functional expression of gastric H,K‐ATPase using the baculovirus expression system

FEBS Letters

De Moel, Mariëlle P.¹  De Caluwé, Godelieve L.J.¹  Van Uem, Tom J.F.¹  De Pont, Jan Joep H.H.M.¹  Swarts, Herman G.P.¹  Klaassen, Corné H.W.¹ 

[1] Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands

关键词: H;    K-ATPase;    Baculovirus expression system;    Sf9 cell;    In vitro expression;    SCH 28080;    H;    K-ATPase (E.C. 3.6.1.36);    magnesium-dependent hydrogen ion transporting and potassium-stimulated adenosine triphosphatase;    SCH 28080 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)imidazo[1;    2a]pyridine;    HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;    CAPS;    cyclohexylamino-1-propanesul-fonic acid;    PAGE;    polyacrylamide gel electrophoresis;    PBS;    phosphate-buffered saline;    Tween 20;    polyoxyethylene sorbitan monolaurate;    PBST;    phosphate-buffered saline;    0.05% Tween 20;    X-gal;    5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside;   

DOI  :  10.1016/0014-5793(93)80237-O

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the α- as well as the β-subunit of the gastric H.K-ATPase. Sf9 cells infected with this virus exhibited a K⁺- and SCH 28080-sensitive ATP-dependent phosphorylation capacity in purified Sf9 membranes similar to Natlve H,K-ATPase. This activity was not present in control membranes containing only one of the two H,K-ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H.K-ATPase.

【 授权许可】

Unknown