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Free keywords:
Abc transporter; Solubilization; Reconstitution; Membrane protein; Photo-crosslinking.; Major histocompatibility complex; Class-ii region; Endoplasmic-reticulum; Rma-s; Mhc; Proteins; Gene; Translocation; Expression; Molecules.; Biochemistry & biophysics.
Abstract:
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 mas analyzed over time, using a non-radioactive crosslinking assay,vith a photo-activateable peptide, in the presence or absence of external lipid, The detergent CHAPS mas found optimally to retain activity and thus allowed us to reconstitute detergent-solubilized, active TAP into proteoliposomes. (C) 1997 Federation of European Biochemical Societies. [References: 35]
