Purification of MBP fusion proteins using engineered DARPin affinity matrix

Abstract

Maltose binding protein (MBP) has a long history as an expression tag with the ability to increase the solubility of fused proteins. A critical step for obtaining a sufficient amount of the MBP fusion protein is purification. Commercially available amylose matrix for the affinity purification of MBP fusion proteins has two main issues: (i) low (micromolar) affinity and (ii) the limited number of uses due to the cleavage of polysaccharide matrix by the amylases, present in the crude cell extract. Here, we present a new affinity purification approach based on the protein-protein interaction. We developed the affinity matrix which contains immobilized Designed Ankyrin Repeat Protein off7 (DARPin off7) – previously identified MBP binder with nanomolar affinity. The functionality of the DARPin affinity matrix was tested on the purification of MBP-tagged green fluorescent protein and flavodoxin. The affinity purification of the MBP fusion proteins, based on the MBP-DARPin off7 interaction, enables the purification of the fusion proteins in a simple two-steps procedure. The DARPin affinity matrix - easy to construct, resistant to amylase, insensitive to maltose contamination, and reusable for multiple purification cycles - provides an alternative approach to commercially available affinity matrices for purification of proteins containing the MBP tag.