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Amylose Affinity Chromatography of MBP 173
properties and high expression levels, the question arises as to why MBP is
not more actively utilized Some of the reasons can be attributed to difficulties
of the early MBP systems and bioprocessing challenges for unwary users
(see Note 4). However, subsequent improvements to the systems have removed
these issues (2). A fundamental problem that still exists with recombinant
protein expression using MBP is that not all MBP fusion constructs work and
the failure rate from a screening expression experiment indicates this could be
as high 30% (28). This percentage is very high for what is one of the best
solubilizing carrier proteins – so why is the percentage so high
Some of the reasons for failure are common to recombinant protein
expression – both cytoplasmic and periplasmic expressions are subject to
the standard E. coli challenges of inclusion body formation and proteolysis,
depending on the growth conditions, host-cell phenotype and physico-chemical
properties of the passenger moiety. With MBP, periplasmic localization can
create an additional challenge as it requires passage through a membrane
and as periplasmic proteins utilize discreet folding machinery, not all MBP
fusion proteins are successfully exported or maintained in the periplasm,
showing significant folding variations or truncations which may or may not
exhibit recombinant protein activity (29,30). Despite these complications, the
major causes for failure appear to be particular to MBP and amylose affinity
chromatography, especially in cases where the fusion protein is present and
soluble but binds inefficiently to the amylose matrix – or even not at all.
There are many reasons why this can occur with MBP and amylose affinity
chromatography and these will now be discussed.
Factors that negatively impact on amylose affinity chromatography can
include buffer additives and cellular biomolecules present in the crude lysis
milieu. Specific problems are noted for the non-ionic detergents Triton X-
100 and Tween 20; New England BioLabs state there is passenger-specific
variability in the ability to bind in the presence of non-ionic detergents (2).
However, it is likely that any additive that can perturb hydrophobic interactions
will be detrimental to amylose affinity chromatography owing to the importance
of aliphatic features of MBP for structure and function and therefore should be
avoided during standard purification (see Note 5 for a further discussions and
guidelines to using additives).
Proteins of the maltose regulon are cellular biomolecules present in the
crude lysis conditions that can potentially affect amylose affinity chromatography.
In the absence of maltodextrins, there is control of protein levels of the
maltose regulon to scavenging levels (18). These basal levels can be elevated
significantly when using alternate carbohydrate sources such as glycerol (as
in terrific broth) or under glucose-limiting growth conditions (as used in a
chemostat or potentially certain bioreactor conditions) (18,31,32). The proteins