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176 Pattenden and Thomas
necessary. If the correct disulphide context is attempted through periplasmic
expression, it is important to omit reducing agents from the buffers. The
standard reducing conditions use 1 mM dithiothreitol (DTT) or 10 mM
-mercaptoethanol in the equilibration, wash and elution buffer.
For amylose affinity chromatography, there are generally three scales.
1. Very small scales, where MBP is used as an affinity group for a magnetic support
for a peptide or protein which acts as a secondary tag (e.g., an antigen) to purify
a completely different biomolecule (e.g., an antibody). This batch mode method
using magnet beads is for small-scale purifications of MBP-passenger protein for
500-μL cell culture supernatant (see Subheading 3.1.).
2. Small-to-moderate scales for protein/peptide study in the laboratory using amylose
agarose or amylose high flow.
3. Larger scales using FPLC apparatus.
Before undertaking purification at moderate or larger scales, a calculation
experiment is advised for optimal purification (see Subheading 3.2.), which
simply approximates the recombinant MBP-passenger protein expression level
for purification. The final yield does not always correlate exactly to the calculation
due to bioprocess variations forming the cleared lysate but provides a
suitable approximation as a starting point. If reliable gel densitometry estimations
can be undertaken, the expression level can be approximated in this
manner by taking a1mL(orsmaller) aliquot of cells when harvesting and
running standard sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS–PAGE) protocols for in-gel protein estimation and by-passing the steps
described in Subheading 3.2. When estimating the amount of matrix, it is
advised to base volumes on 3 mg/mL binding capacity unless a1mLpilot
experiment is conducted for further optimization (most appropriate when larger
scales are attempted).
The batch and semi-batch method is principally for smaller scale purifications
of MBP-passenger protein using cell culture supernatant volumes as low as
500 μL. The method can be applied at moderate scales with very good success
where an FPLC system is not available. The critical parameter limiting the
scale is liquid handling associated with the matrix, especially where the MBPpassenger
protein has a lower binding capacity (∼3 mg/mL); more matrix is
needed and can often result in clogging and flow restrictions at high protein
loads. Flow restrictions can also occur using the agarose matrix and large
liquid volumes when a semi-batch column approach is used under gravity. The
flow, using columns up to 2 mL volumes can be enhanced during loading
and washing steps using a vacuum manifold (e.g., a ‘piglet’), especially when
using the amylose high flow matrix where an FPLC system is not available and
simple PD10 disposable columns (BioRad) work well using such manifolds.