Figure_1.tif (2.67 MB)

rIDE is expressed in insect cells, degrades insulin, and binds to VZV gE.

figure

posted on 2013-02-21, 00:48 authored by Qingxue Li, Mir A. Ali, Kening Wang, Dean Sayre, Frederick G. Hamel, Elizabeth R. Fischer, Robert G. Bennett, Jeffrey I. Cohen

(A) Structure of HA-tagged rIDE and its promoter in recombinant baculovirus. (B) rIDE was purified, eluted under native conditions, and stained with Coomassie Blue. Soluble rIDE is expressed as a 110 kDa protein in insect cells. (C) HPLC profiles of insulin degradation fragments observed after co-incubation of radiolabeled insulin with rIDE expressed in insect cells, endogenous IDE extracted from rat liver (top panel), or recombinant IDE (6His/Flag-IDE) expressed in eukaryotic cells (bottom panel). (D) Insulin-degrading enzymatic activities of IDE proteins. (E) ELISA assay for binding of rIDE to gEt, gEt lacking the IDE binding domain (gEtΔ32-71), gEt defective in binding to VZV gI (gEtΔ163-208), and a negative control protein (P7.5, vaccinia 7.5 protein). The horizontal dotted line indicates the level of nonspecific binding based on the control protein.