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15. Cells can be pelleted from the aliquot by centrifugation (6000 × g, 15 min, 4°C).
Ensure the supernatant is decanted completely. It is optional to store the cell
pellet and process at a later date. Cell pellets should not be stored at 4°C, but may
be stored for several weeks at –20°C before proceeding. Longer term storage
should be at –80°C or under liquid nitrogen. If stored frozen, thaw the pellet in
ice water when ready to proceed.
16. New England BioLabs state amylose resin can withstand centrifugation at up to
6000 × g (2).
17. Transferring to a new vessel during washing steps decreases non-specific contaminants
that adhere to vessel walls or remain in vessels and carry over to subsequent
steps.
18. For amylose agarose, the total protein concentration should be ≤2.5 mg/mL for
best binding to the amylose matrix with reduced viscosity (which causes severe
flow restrictions). The dilution to 2.5 mg/mL restricts the batch mode by what
volume can be handled in terms of the size of centrifuge tubes that can be used
and column size. For batch and semi-batch modes, no more than 5 mL matrix is
recommended.
19. The resin may be reused up to 10 times (2), but caution should be made if
regenerating at 4°C as SDS can precipitate over time.
20. When using a dialysis cassette, it is necessary to cut away the membrane window
using a scalpel to properly extract the amylose matrix to avoid foaming.
References
1. Maina, C. V., Riggs, P. D., Grandea, A. G., III, Slatko, B. E., Moran, L. S.,
Tagliamonte, J. A., McReynolds, L. A., and Guan, C. D., (1988), An Escherichia
coli vector to express and purify foreign proteins by fusion to and separation from
maltose-binding protein, Gene 74, 365.
2. pMAL Protein Fusion and Purification System (Expression and Purification of
Proteins and Cloned Genes), (2006), New England Biolabs. V 5.1.
3. Addgene, (2006), http://www.addgene.org.
4. Fox, J. D., Routzahn, K. M., Bucher, M. H., and Waugh, D. S., (2003),
Maltodextrin-binding proteins from diverse bacteria and archaea are potent
solubility enhancers, FEBS Lett. 537, 53.
5. Nallamsetty, S., Austin, B. P., Penrose, K. J., and Waugh, D. S., (2005), Gateway
vectors for the production of combinatorially-tagged His6-MBP fusion proteins in
the cytoplasm and periplasm of Escherichia coli, Protein Sci. 14, 2964.
6. Luecke, H., and Quiocho, F. A., (1990), High specificity of a phosphate transport
protein determined by hydrogen bonds, Nature 347, 402–406.
7. Pflugrath, J. W., and Quiocho, F. A., (1988), The 2 A resolution structure of the
sulfate-binding protein involved in active transport in Salmonella typhimurium, J.
Mol. Biol. 200, 163–180.
8. de Pina, K., Navarro, C., McWalter, L., Boxer, D. H., Price, N. C., Kelly, S. M.,
Mandrand-Berthelot, M. A., and Wu, L. F., (1995), Purification and characteri-