Peptide binding and photo-crosslinking to detergent solubilized and to reconstituted transporter associated with antigen processing (TAP).
The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally active, heterologously expressed human TAP from microsomal membranes. The efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions. The activity of the solubilized TAP complex was analyzed over time, using a non-radioactive crosslinking assay with a photo-activateable peptide, in the presence or absence of external lipid. The detergent CHAPS was found optimally to retain activity and thus allowed us to reconstitute detergent-solubilized, active TAP into proteoliposomes.
Uebel
S
S
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Plantinga
T
T
Weber
P J
PJ
Beck-Sickinger
A G
AG
Tampé
R
R
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D003902:Detergents; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008285:Major Histocompatibility Complex; D008861:Microsomes / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010777:Photochemistry; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D014162:Transfection
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D003432:Cross-Linking Reagents; D003902:Detergents; D015395:Histocompatibility Antigens Class I; D008081:Liposomes; D010455:Peptides; D011510:Proteolipids; D011994:Recombinant Proteins; C073119:TAP1 protein, human; C020485:proteoliposomes