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Amylose Affinity Chromatography of MBP 183
differences in equilibrium constants are reflected in different dissociation rates
(K d ∼3.5 μM, maltose; ∼0.16 μM, maltotriose) (36).
2. Three amylose affinity chromatography matrices are manufactured by New
England BioLabs, being functionalized onto magnetic beads, agarose and a high
flowing support matrix, though a custom matrix can be manufactured (40).
Amylose magnetic beads have a binding capacity of up to 10 μg/mg (supplied as
a 10 mg/mL suspension). Amylose agarose has a binding capacity of 3 mg/mL
for MBP and 6 mg/mL for an MBP--galactosidase protein. The typical flow
velocity of the amylose resin is 1 mL/min in a 2.5 cm × 10 cm column, and
the matrix can withstand small manifold vacuums (e.g., a “piglet”). The amylose
matrix can suffer from flow restrictions, and so total protein loading should be
≤2.5 mg/mL. Amylose high flow has a binding capacity of approximately 7
mg/mL for an MBP-paramyosin protein. The exact chemical nature of the matrix
is not described but has a pressure limit of 0.5 MPa (75 psi), a maximum flow
velocity of 300 cm/h, and recommended velocities are below 60 cm/h being
10–25 mL/min (for 1.6-cm and ∅2.5-cm columns respectively).
3. New England BioLabs provide simple lysis conditions to access MBP-passenger
proteins located in the periplasm (2). The method involves lysis using sucrose,
EDTA and MgSO 4 and low speed centrifugation. This is an effective means
of purification as the periplasmic MBP-mediated transport system is susceptible
to mild osmotic shock, causing the loss of transport activity and recovery of
periplasmic-binding proteins in the osmotic medium (18).
4. When cloned using Eco RI, early systems would not be cleaved by Factor Xa
and some constructs produced Factor Xa sites that were inefficiently cleaved –
likely due to structural complications induced about the cleavage site. In general,
the Factor Xa bioprocessing is also unfavoured by many users owing to the
promiscuity of Factor Xa; it is well documented that Factor Xa cleaves noncanonical
sites of the desired recombinant passenger protein in regions that
contain arginine at the P1 site, possibly where regions are in proteolytically
preferred extended conformations (41). Methods to reversibly acylate such sites
have been described (42–44), but in the hands of these authors such methods
are ineffective. Amylose was originally functionalized onto agarose and had
earlier been reported to have a binding capacity of >3 mg/mL, which has been
revised (see Note 2). This seemed to be relatively low compared to other affinity
purification systems and had a tendency to encounter viscosity problems at
concentrated protein loadings, causing the columns to suffer flow restrictions and
creating a need to work with dilute loadings (thereby imposing liquid handling and
chromatographic scale limitations). Generally, the range of improved constructs,
diversity in protease sites and development of the amylose high flow matrix
overcome all these issues when a bioprocess is properly planned with the physicochemical
properties of the passenger protein or peptide carefully considered.
5. Where a detergent is required for the passenger protein to remain soluble, it is
advisable to utilize the additive in buffers following amylose affinity chromatography
or in the elution buffer. Where this is not possible, as a general rule, the