Interaction studies of the E1- and E2-domain.
(a) Analytical gel filtration using the constructs APP-E1_ED_AcD and APP-E2_JMR. The UV-traces of the GPC analysis of the isolated and mixed proteins are given as indicated on top of the panel. For clarity the baseline is shifted arbitrarily. Upon mixing no increase of the molecular weight was observed, indicating the lack of a strong interaction between both constructs. (b) Pull-down assay with APP-E1_ED_AcD (His-tagged) and APP-E2_JMR (no His-tag) using Ni-NTA material. After centrifugation, APP-E2_JMR remains completely in the supernatant (soluble) and only His-tagged APP-E1_ED_AcD can be eluted. The first four lanes show that APP-E2_JMR (no His-tag) does not bind unspecifically to Ni-NTA (control). The sizes of the molecular weight (MW) marker bands are given on the left of the gel in kDa. (c) The pull-down assay with the constructs APP-E1 (His-tagged) and APP-E2 (no His-tag) demonstrates no interaction and shows that the flexible linker has no influence on a potential interaction of the domains. (d) Pull-down assay in the presence of low molecular weight heparin (10-12 sugar rings) indicates that heparin does not influence an interaction of APP-E1_ED_AcD and APP-E2_JMR.