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Amylose Affinity Chromatography of MBP 171
ligands in the environment through integral membrane proteins, Gram negative
organisms have two membranes separated by the periplasmic space, presenting
a challenge to co-ordinate the uptake, movement and translocation across these
diverse structural features. In native E. coli, MBP mediate processes by acting as
a chemoreceptor for -(1→4)-D-glucose polysaccharides (maltodextrins) (18);
the binding of the ligand induces a conformational change in MBP that allows
the selective recognition by specific integral membrane proteins, receptors and
porins for the following:
1. Chemotaxis by inner membrane receptors: Maltose chemotaxis is the process by
which the bacteria move in response to a maltodextrin concentration gradient
through signals that are transmitted to the flagellar.
2. Transport of maltodextrins: Firstly by porins of the outer membrane, raising the
periplasmic concentration of the maltodextrins and subsequent energy-dependent
active translocation of maltodextrins into the cytoplasm by integral membrane
proteins.
The proteins involved in maltodextrin chemotaxis and transport are collectively
termed the maltose regulon of E. coli (18). In order to mediate the
separate processes of chemotaxis and transport, MBP is normally present in
a very large (∼50 fold) excess compared to the associated membrane protein
components of the maltose regulon. Another suggestion for the high levels of
MBP is that MBP has molecular chaperone properties that may help in protein
folding and renaturation in the periplasm (19,20). There are two ways by which
MBP could be involved in protein folding. One is passive – by being a stable
and readily ‘foldable’ protein that is attached to the desired recombinant protein
(21,22). Alternatively, MBP has been hypothesized to actively refold proteins –
through interactions at hydrophobic regions of MBP (19,20), possibly with the
hydrophobic surface clusters important for interacting with proteins involved
in maltodextrin chemotaxis and transport (23,24).
MBP mediates diverse cellular responses for maltodextrin metabolism in
the presence of any -(1→4)-D-glucose polysaccharide of up to 8 glucose
units in length. Maltose binds to MBP with the glucose ring oxygen atoms
all on the same side, and adopting this correct conformation for alignment of
hydrogen bonding interactions within MBP is critical for affinity. Amylose
is essentially a repeating maltose polymer with flexible polysaccharide chains
joined by the -(1→4) links. Amylose affinity chromatography exploits the
maltodextrin-like affinity of MBP as the basis for purification (see Note 2 for
matrix properties and chromatography conditions).
Structural aspects of MBP are important for amylose affinity chromatography.
The polypeptide chain of MBP is present as two globular domains,
and the maltodextrin-like ligands bind within a ligand-binding cleft located
at an interface formed by the two globular domains (25). Essentially, MBP