Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells

FEBS Letters

van Endert, Peter M.¹  Tampé, Robert²  Ehring, Bettina²  Uebel, Stephan²  Meyer, Thomas H.² 

[1] INSERM U25, Hospital Necker, 161 rue de Sevres, 75743 Paris Cedex 15, France;Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany

关键词: ABC transporter;    Membrane protein;    Traffic ATPase;    Antigen presentation;    Peptide transport;    Baculovirus;    Insect cell;    ABC;    ATP binding cassette;    AMP-PNP;    5′-adenylylimidodiphospate;    BV;    baculovirus;    DM;    dodecyl-β-d-maltoside;    DOTAP;    N-[1-(2;    3-dioleoyloxy)propyl]-N;    N;    N-trimethylammoniummethylsulfate;    IMAC;    immobilized metal ion affinity chromatography;    mAb;    monoclonal antibody;    Ni-IDA;    nickel iminodiacetic acid;    pAb;    polyclonal antibody;    p.i.;    post infection;    TAP;    transporter associated with antigen processing;    wt;    wild-type;   

DOI  :  10.1016/0014-5793(94)00908-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Using the baculovirus expression system the gene products of human tap1 and tap2 were over-expressed as wild-type as well as oligohistidine fusion proteins in Spodoptera frugiperda (Sf9) insect cells. Both gene products were co-expressed within the same cells and were found enriched in microsomal membranes. Immunoprecipitation and immobilized metal affinity chromatography revealed complex formation between TAP1 and TAP2. The expressed TAP complex was shown to be functional by peptide translocation into microsomes of Sf9 cells. Peptide transport strictly requires TAP1 and TAP2 as well as ATP. For the first time the functional expression of the human TAP complex in insect cells has been demonstrated, indicating that additional cofactors of a highly developed immune system are not essential for peptide transport across microsomal membranes.

【 授权许可】

Unknown