Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells.
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.
Meyer
T H
TH
Max-Planck-Institut für Biochemie, Martinsried, Germany.
van Endert
P M
PM
Uebel
S
S
Ehring
B
B
Tampé
R
R
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D011233:Precipitin Tests
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; C073119:TAP1 protein, human; C071692:TAP2 protein, human