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Abstract

Immobilized metal ion affinity chromatography has shown promise for isolating desired proteins from host cell extracts, based on differential affinities for chelated metal ions. This article focuses on modeling the effect of loading pH on the purity and binding capacity of target proteins, during their separation from the cell extract of Escherichia coli, using immobilized copper affinity chromatography. Early chromatography experiments revealed that cellular proteins of E. coli elute in two separate peaks from an immobilized copper column under a decreasing pH step gradient. Thus, the cell extract is modeled by a mixture of two proteins, tuna heart cytochrome c and chicken egg white lysozyme. Transport and binding parameters of these proteins are evaluated over a wide pH range and used in a mathematical model of multicomponent chromatography. Target proteins are chosen such that they elute at a pH between the elution pH of cytochrome and lysozyme. Simulation results showed that decreasing the loading pH to a value between the elution pH of the weaker copper binding proteins of E. coli and that of the target protein may not only decrease the amount of E. coli proteins bound in the column but lead to a substantially higher binding capacity for the target protein.