Controlling the orientation of immobilized proteins on an affinity membrane through chelation of a histidine tag to a chitosan-Ni++ surface

The ability of histidine-tagged proteins to chelate to Ni++ ions that are coordinated to functionalized polymeric matrices is the basis of affinity column separations. In this study, affinity membranes based on a Ni++-chelating chitosan surface were fabricated to immobilize C-terminus hexahistidine-tagged green fluorescent protein (his-GFP). The binding of GFP antibody (antiGFP) to the immobilized his-GFP was measured and compared to a membrane with a chitosan surface to which his-GFP was immobilized through amine-glutaraldehyde chemistry as a control. Both membranes had comparable amounts of his-GFP immobilized on the surface. However, the amount of antiGFP bound to the Ni++-chelated his-GFP at saturation was higher than that bound to the glutaraldehyde-immobilized his-GFP by a factor of five. Furthermore, fitting the data to a single-site Langmuir model resulted in an affinity constant for the Ni++-chelated his-GFP towards antiGFP that was 14 times higher than the glutaraldehyde-immobilized his-GFP. The higher affinity suggests that immobilizing a protein at its C-terminus results in the proper orientation for subsequent antibody binding. At low antibody concentrations, the sensitivity of the affinity membrane is 70 times that of the control.