Enhancement of binding activity of soluble human CD40 to CD40 ligand through incorporation of an isoleucine zipper motif
Abstract
Aim: To investigate the effect of incorporation of an isoleucine zipper (IZ) motif into 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 Escherichia coli. The recombinant proteins were purified to homogeneity after refolding from inclusion bodies. Their molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography, and their binding activity 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 did shCD40His. Furthermore, size-exclusion chromatography demonstrated that shCD40IZ existed in high molecular mass forms that were most likely to be trimers in solution.
Conclusion: Incorporation of an IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.
Keywords:
Methods: Prokaryotic expression vectors for 2 soluble CD40 derivatives, shCD40His and shCD40IZ containing an IZ dowain, were constructed and expressed in Escherichia coli. The recombinant proteins were purified to homogeneity after refolding from inclusion bodies. Their molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography, and their binding activity 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 did shCD40His. Furthermore, size-exclusion chromatography demonstrated that shCD40IZ existed in high molecular mass forms that were most likely to be trimers in solution.
Conclusion: Incorporation of an IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.