Adsorption of pure recombinant MBP-fusion proteins on amylose affinity membranes

The specific interaction between MBP-fusion proteins and amylose based affinity membranes has been presented recently. In this work, the attention is focused on the influence of ionic strength on the equilibrium isotherm and on the kinetics of adsorption and desorption. Three different MBP-fusion proteins have been used: MBP-rubredoxin (MW 51 kDa), MBP-intein-CBD (MW 97 kDa) and MBP-beta galactosidase (MW 160 kDa) characterized by different dimensions. The equilibrium data follow the Langmuir isotherm, whose parameters q(m) and K-d show a dependence on salt concentration in the feed solution. By increasing the ionic strength, the binding capacity at saturation q(m) decreases while the equilibrium constant K-d increases, indicating that a higher concentration of NaCl reduces the affinity interaction between protein and ligand. A simple kinetic model has been investigated for the interpolation of adsorption and elution curves. No mass transfer effects have been considered within the liquid phase and inside the pores of the stationary phase. The high processing speed of membrane affinity filtration has been exploited. The kinetics of adsorption and elution have been described and the values of the kinetic constants have been obtained both for adsorption and elution conditions for all proteins. The influence of the action of a competing substrate like maltose on the removal of the immobilized product has been also determined. (c) 2005 Elsevier B.V. All rights reserved.