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

Abstract

Using the baculovirus expression system the gene products of human tapl and tap2 were over-expressed as wild-type as well as oligohistidine fusion proteins in Spodopterafrugiperda (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 ATE For the first time the functional expression ofthe 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.

TAP and TAP-like — Brothers in arms?

“…Therefore, TAPL can be energized by different nucleoside triphosphates with a slight predilection for purine based nucleotides. This broad specificity is also found with other ABC transporters (Alshawi and Senior 1993;Meyer et al 1994;Müller et al 1994) and results from only a π−π interaction between the base of the nucleotide with the NBD (Hung et al 1998). …”

“…This heterodimeric transport complex consists of TAP1 (ABCB2) and TAP2 (ABCB3) (Kleijmeer et al 1992;Lacaille and Androlewicz 1998). Both subunits are essential and sufficient for antigen processing (Powis et al 1991;Spies and DeMars 1991;Meyer et al 1994). …”

Intracellular peptide transporters in human – compartmentalization of the “peptidome”

“…This indicates that TAPL translocates peptides into a post-ER compartment for accumulation. In contrast, TAP transports peptides into the ER lumen, where they are retro-translocated via the SEC complex [46], if not trapped by N-core glycosylation [61,65]. In stably transfected SKOV3 cells, TAPL was found to be co-localized with the lysosome-associated membrane Fig.…”

“…Peptide binding is ATP-independent [88,90], whereas, peptide translocation requires ATP hydrolysis [3,61,65,79]. Peptide binding is composed of a fast association step followed by a slow structural reorganization of the TAP complex [68].…”

Gene transfer of human interferon gamma complementary DNA into a renal cell carcinoma line enhances MHC-restricted cytotoxic T lymphocyte recognition but suppresses non-MHC-restricted effector cell activity

“…Immunodetection of LMP and TAP proteins was performed using enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, UK) according to the manufacturer's instruction. For the detection of TAP1 and TAP2 proteins, the membranes were incubated overnight at room temperature with either anti-TAP1 mAb 148.3 (1:10 dilution of hybridoma supernatant) 68 or anti-TAP2 mAb 429.3 (1:20 dilution of hybridoma supernatant), 69 followed by a 1 h incubation with a 1:8000 dilution of horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (A4416; Sigma) at room temperature. After ECL detection the membrane was stripped of bound antibodies by incubation in a solution containing 60 mm Tris-HCl, 2% SDS, 100 mm ␤-mercaptoethanol, pH 6.7 at 50°C for 30 min.…”