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174 Pattenden and Thomas
of the maltose regulon and cellular inducers of the regulon are particularly
detrimental as they can bind and/or modify the amylose matrix directly
or sequentially, often releasing maltose, maltotriose or analogues as a byproduct
which can elute MBP fusion proteins from the amylose matrix.
These proteins include maltodextrinyl-specific, phosphorylases, transferases,
glucosidases, -cyclodextrinases, transacetylases, periplasmic and cytoplasmic
-amylases and amylase-like enzymes (18), and these proteins are likely the
cause of deterioration of amylose affinity matrices (see Note 6). However, the
basal scavenging levels can be maintained with high glucose concentrations that
exert strong catabolite repression to the maltose regulon and maltodextrinylspecific
operons (18). A D-glucose concentration of 0.2% is sufficient in Lauria
Bertani media to suitably suppress unwanted protein expression including
leaky expression of the MBP fusion protein (2), but the concentration of the
suppressor will alter depending on media types and growth parameters, such
as growth densities and the phase of growth. Leaky expression from pMAL
vectors is also controlled by the presence of glucose which ensures the tac
promoter is not induced in the absence of isopropyl -D-thiogalactopyranoside.
Another possible cause of purification failure comes from the proposal that
certain chaperone-like interactions may be detrimental if constructs form soluble
aggregates through physical association, becoming trapped in a folding intermediate
state such that the MBP-passenger protein forms a stable globular form
termed a sequestered intermediate (19). However, it is important to consider the
nature of the cloning at the C terminus, which is close to the ligand-binding cleft
and the hinge region. It is possible that excessive removal of nucleotides during
cloning will shorten the linker regions introduced with more recent constructs
from New England BioLabs, and some constructs may interact with the ligandbinding
cleft depending on their physical properties; this may disrupt ligand
binding or important hinge movements that are necessary for high affinity
binding of the amylose matrix leading to a failure of purification. It is also
important to understand detrimental interactions for purification need not occur
locally (in cis or upon the same MBP-passenger molecule), but could also
arise from multiple regions of the MBP-passenger protein interacting in trans
on neighbouring MBP-passenger molecules or with other biomolecules in the
purification milieu. In such a scenario, the involvement of hydrophobic regions
in or about the substrate-binding cleft may occlude binding to the amylose
matrix or involve the hinge region and thereby impede normal conformational
changes necessary for binding to the amylose matrix. Therefore, the purification
failure of some MBP-passenger proteins could be exacerbated by molecular
crowding as a consequence of high protein expression levels, the growth
conditions for expression or protein concentrations when lysis is conducted
in small volumes. It should also be noted that detrimental protein–protein