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170 Pattenden and Thomas
1. Introduction
Affinity chromatography of maltose-binding protein (MBP) (1) exploits the
binding of amylose that is functionalized as the stationary phase to magnetic
beads, agarose or an inert matrix. As for other forms of affinity chromatography,
the successful purification using amylose affinity chromatography is critically
linked to an intimate understanding of the biomolecular interactions (and
complications) that can occur due to the specific and unique features of MBP
and amylose biology. Likewise a limitation to broader applications, greater
developments and the reason for misunderstandings that arise with regard to
MBP and amylose affinity chromatography are the failures to completely grasp
and exploit aspects of this biology. This chapter highlights the facets of MBP
and amylose biology and chemistry that are relevant to affinity purification and
discusses how these facets negatively impact purification or can be exploited
to achieve purification. Methods are presented for native purification in batch,
semi-batch and fast protein liquid chromatography (FPLC) modes, and a new
matrix-assisted dialysis refolding method is described that is suitable for batch
and semi-batch modes.
MBP, also referred to as maltodextrin-binding protein (and sometimes
written unhyphenated), can be expressed at arguably the highest levels for
any recombinant carrier protein. Developed by New England BioLabs from
Escherichia coli, there are now six constructs commercially available for
periplasmic or cytoplasmic expression with a Factor Xa, Genenase I or
Enterokinase protease site engineered into the constructs (2). There is also
a series of MBP constructs developed by David S Waugh (National Cancer
Institute at Frederick) that can be obtained from the non-profit distributor of
biological reagents AddGene (3,4,5) which include Gateway© His6-MBP and
non-E. coli-sourced MBP, typically using a TEV protease site (tobacco etch
virus nuclear inclusion protease site) (4,5). New England BioLabs also provide
a range of suitable E. coli host cells that are useful for MBP expression free
of charge and have very reasonable licensing and royalty terms, making MBPbased
recombinant carrier protein expression and purification also one of the
most economically achievable affinity chromatography systems available for
both research and commercial ventures.
MBP belongs to the bacterial superfamily of periplasmic-binding proteins
that are monomeric bilobular proteins with molecular weights in the range
of 25–45 kDa, containing a single ligand-binding site with micromolar dissociation
constants for diverse ligands ranging from ions (6–9), amino acids
(10–13), oligopeptides (14,15) and carbohydrates (16,17) (see Note 1 for MBP
biophysical properties). Within Gram negative bacteria, the periplasmic-binding
proteins are involved in the chemotaxis and transport of their respective ligands.
Unlike Gram positive bacterium that directly sense and responds to specific