Figure 3. Soluble TAPBPR loop variants exhibit reduced ability to mediate peptide exchange on surface HLA-A*68:02 molecules.

(a) Expression and purity of soluble forms of WT, L30G, ØG30L, and Øloop TAPBPR variants after their purification from the culture supernatant using Ni-affinity. (b) Differential scanning fluorimetry demonstrates the three TAPBPR loop mutants have equivalent thermal denaturation profiles as TAPBPR^WT. (c) Histograms of the typical fluorescent peptide binding to IFNγ-treated HeLaM cells incubated -/+100 nM exogenous soluble TAPBPR variant for 15 min at 37°C, followed by incubation with 10 nM ETVSK*QSNV or YVVPFVAK*V for an additional 15 min. TAPBPR^TN5, in which isoleucine at position 261 is mutated to lysine, to produce a TAPBPR variant which cannot bind to MHC I, is included as a control (d) Bar graphs show the reproducibility of results in (c). (e) Dose response curves of fluorescent peptide binding to IFN-γ treated HeLaM cells incubated with increasing concentrations of the soluble TAPBPR variants prior to the addition of 10 nM ETVSK*QSNV. Error bars represent MFI -/+SD from four independent experiments. ****p≤0.0001 using unpaired two-tailed t-tests.