Purification of MBP-β-galactosidase and MBP-rubredoxin through affinity membrane separation

Abstract

A system based on the use of affinity membranes for the recovery and purification of a class of fusion proteins containing the Maltose Binding Protein (MPB) domain has been studied. An affinity support was obtained through a chemical modification protocol of microporous cellulosic membranes, and amylose was used as the specific ligand. A membrane module was realized in a column configuration, suitable for flat sheet membranes. The total membrane area available was arranged in a series of stages in order to benefit of the fluid-dynamic effects throughout the entire stack. The performance of the process was compared with the one offered by stationary phases based on porous beads or resins. Two different fusion proteins, MBP-β galactosidase and MBP-rubredoxin, characterized by a molecular weight of 160 and 51 kDa, respectively, were used. The feasibility of a single-step separation process of MBP fusions with amylose affinity membranes has been demonstrated, with good results both in terms of selectivity and purity of the recovered product. In comparison to the commercially available supports: (i) the binding capacity per unit volume of the membranes obtained is approximately the same; (ii) the process time is much shorter when affinity membranes are used; and (iii) protein concentration as well as purity of the resulting protein solutions are higher for the affinity membrane process.

Acknowledgments

The contribution of Dr. Cristiana Boi, University of Bologna, and Prof. Georges Belfort, Rensselaer Polytechnic Institute, in initiating this work is gratefully acknowledged. Fruitful discussions with Prof. Alessandro Hockoeppler, University of Bologna, are also gratefully acknowledged.