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Amylose Affinity Chromatography of MBP 175
interactions can be independent of size, and the authors have found amylose
affinity chromatography can fail even with small peptides of 4 kDa attached
to MBP.
Though currently some mechanisms are only hypotheses, approaches to
address these problems can result in successful purification following failure,
and the overall issue has also been approached using additional accessory
tags (33,34). The authors have found it is very speculative to try to consider
the three-dimensional topology of the passenger moiety and MBP in stereo
and so have developed a novel matrix-assisted dialysis refolding method
(see Subheading 3.6.) that is useful for troubleshooting purifications that
have failed as well as a general means for purification of recombinant MBPpassenger
proteins that can refold in light of the failure of conventional methods.
The matrix-assisted dialysis refolding method is essentially refolding denatured
MBP-passenger protein within a dialysis cassette or membrane in the presence
of the amylose resin. Refolding in the presence of the amylose ligand can allow
the MBP-passenger protein to refold attached to the matrix (as the binding cleft
forms around the ligand) allowing capture. We have found the contaminants in
the resin from denatured debris did not carry over as significantly as imagined,
and other refolding conditions will no doubt be successful.
1.2. Amylose Affinity Chromatography
As MBP is active over a wide pH and salt range, there are many choices
for buffer conditions that can be used, but generally buffers around a neutral
slight basic nature (7.5–8) with modest ionic strengths (100–500 mM) are
best. Because MBP has an acidic isoelectric point (pI) (see Note 1), when
concentrated it can affect the pH of the solution and so it is recommended to
use an appropriate strength of the buffer (>20 mM). When deciding on the
exact buffer composition, it is important to consider the overall bioprocess
(including lysis conditions and downstream processes such as proteolytic tag
removal and secondary chromatography) and to formulate the buffer to interface
with these other processes. For example, if a Factor Xa cleavage is necessary,
certain protease inhibitors (see Note 7) and ethylene glycol tetraacetic acid
(EGTA) (see Note 8) are not desired in wash buffers or elution buffers or need
to be thoroughly removed before eluting the protein. Protease inhibitors and
metal chelating agents are compatible with all matrices used (e.g., Leupeptin,
Aprotinin, Pepstatin, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic
acid (EDTA) and EGTA).
It is also important to consider the disulphide context that may be required.
In general, the buffers for amylose affinity chromatography can include redox,
oxidizing or reducing agents to either maintain or break disulphide bonds as