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Aim:To investigate the effect of incorporation of an isoleucine zipper(IZ)motifinto CD40 on binding activity of CD40 for the CD40 ligand(CD40L).Methods:Prokaryotic expression vectors for 2 soluble CD40 derivatives,shCD40His andshCD40IZ containing an IZ dowain,were constructed and expressed in Escheri-chia coli.The recombinant proteins were purified to homogeneity after refoldingfrom inclusion bodies.Their molecular weights in solution of shCD40His andshCD40IZ were compared by size-exclusion chromatography,and their bindingactivity for CD40L on Jurkat T cells was determined by flow cytometry.Results:shCD40His and shCD40IZ were generated.Both of them possessed significantbinding activity for the cognate ligand CD40L expressed on the cell surface.shCD40IZ had much higher binding activity to its ligand(CD40L)than didshCD40His.Furthermore,size-exclusion chromatography demonstrated thatshCD40IZ existed in high molecular mass forms that were most likely to be trimersin solution.Conclusion:Incorporation of an IZ motif into CD40 enhances itsbinding activity for CD40L through trimerization of the CD40 derivative.
Aim: To investigate the effect of incorporation of an isoleucine zipper (IZ) motifinto CD40 on binding activity of CD40 for the CD40 ligand (CD40L). Methods: Prokaryotic expression vectors for 2 soluble CD40 derivatives, shCD40His and shCD40IZ containing an IZ dowain, were constructed and expressed in Escheri-chia coli. The recombinant proteins were purified to homogeneity after refolding from inclusion bodies. The molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography, and their bindingactivity for CD40L on Jurkat T cells was determined by flow cytometry . Results: shCD40His and shCD40IZ were generated ..Both of them possessed significant binding activity for the cognate ligand CD40L expressed on the cell surface. ShCD40IZ had much higher binding activity to its ligand (CD40L) than didshCD40His.Furthermore, size-exclusion chromatography demonstrated thatshCD40IZ existed in high molecular mass forms that were most likely to be trimersin solution. Confluence: Inc or of of IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.