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Amylose Affinity Chromatography of MBP 185
9. When preparing all buffers add ingredients making the buffer to 90% of final
volume and titrate the pH using HCl to the desired concentration, making up
to the final volume. With urea-containing buffers, dilute dry ingredients to 50%
of final volume and fully dissolve the solids. Urea dissolves in an endothermic
reaction (turning the solution cold), therefore, allow the buffer solution to return
to room temperature once fully dissolved before making to 90% of the final
volume and adjusting the pH with HCl.
10. The different amylose matrices are supplied by New England BioLabs in a
20% ethanol solution that can negatively influence purification and requires
removal. The amylose magnetic beads are supplied with 0.05% Tween-20 that
can be significant at very small protein concentrations. In related applications,
the authors have found that low levels of residual detergents (especially from
regeneration solutions) can still remain and have found SDS and detergent mixed
micelles particularly difficult to remove. The authors have analyzed removal
of detergent and mixed micelles using surface plasmon resonance (BIAcore
T100, BIAcore) and dual polarization interferometry (AnaLight 200, Farfield
Instruments), finding dilute methanol-containing solutions are most efficient for
removal of these agents.
11. The manner of lysis is dependent on available equipment, scale, bacterial
strain (which may encode a lysozyme in the case of pLysS strains (45)) and
whether periplasmic (see Note 3) or cytoplasmic expression is undertaken.
For cytoplasmic expression, mechanical lysis is more effective for successful
MBP purification than chemical lysis as chemical lysis employs agents that
are frequently incompatible with MBP chemistry (see Note 5). Large scales
may require a cell disruptor such as a French press to successfully achieve
lysis and suitable viscosity, whereas moderate and small scales may employ
sonication. Small-scale lysis can also be achieved using standard freeze-thaw
cycling techniques but may have elevated viscosities due to intact nucleic acid
being present. The standard method of the authors employs sonication on ice
using a Branson B30 Sonifier at 70% duty cycle to 20 kHz (∼5.5 output but
varies with turbidity), for 3 min with 3 × 30-s bursts with rests between cycles.
Typically, the authors form a cleared lysate by centrifugation at 45,700 × g for
45 min at 4ºC.
12. It is important to avoid vigorous mixing during all liquid handling steps as this
causes loss of product by denaturation (foaming) of protein solutions. For this
reason, liquid handling and mixing is conducted gently.
13. If suitable mixers or cold rooms are unavailable, place the suspension on ice
and invert gently every 5 min. The binding reaction is enhanced by collisions
between amylose and MBP species as opposed to passive diffusion and therefore
gentle agitation of the suspension maximizes the capture to the amylose matrix.
14. Higher concentrations of maltose (50–100 mM) can influence storage of eluted
protein samples as a cryoprotectant, and so, eluted samples sometimes require
dilution below 20 mM for proper freezing.