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IMAC of Histidine-Tagged Fusion Proteins 147
non-specific interactions that may occur because of excess stationary support not
interacting with the target molecule are addressed through the proposed stringent
pre-equilibration, equilibration and washing regimens.
6. In these instances, significant metal leaching may occur during loading, reducing
the capacity of the Ni-NTA but not below 1 mg of protein per ml of Ni-NTA.
7. If monitoring A 280 nm note that imidazole absorbs at this wavelength and so
achieving baseline should only be relative to the absorbance of the equilibration
buffer at A 280 nm . In wash and elution steps, care should be taken to avoid confusing
an increasing A 280 nm signal due to the use of a higher imidazole concentration
with that of elution of a protein.
8. Not all supports should be stored charged with metal ions. Silica-based supports
should be stored free of metal ion and only charged when required. The charged
metal ion causes a localized low pH microenvironment that can damage these
supports over time, decreasing the life expectancy of the column.
9. Metal ions that could be used for this work are preferably the hard Lewis metal
ions such as Fe 3+ and any of the lanthanides. Hard Lewis metal ions such as Ca 2+
could also be used; however, a good chelating stationary phase to use this metal ion
in IMAC for the purification of proteins does not exist commercially. Al 3+ is also
another example; however, the commercially available 8-hydroxyquinoline support
would be more useful over IDA stationary phases for this metal ion. Borderline
Lewis metal ions like Cu 2+ and Co 2+ can also be used in this mode (24,25).
10. In this way, insight will be gained as to the mode of binding of the target protein.
If the protein is recovered in this step, then the binding is mediated by histidine
binding to the IMCC. If not, then the protein is bound in a non-specific manner,
such as hydrophobic interaction with the spacer arm of the ligand.
11. It is known from attempting the steps described in Subheading 3.1 that the target
protein remains bound in the presence of 0.2 M imidazole + 0.5 M NaCl. Loading
under more stringent conditions may assist later elution by reducing the number
of binding modes available to the protein. Higher binding stringency may also
improve product purity and column capacity, as less binding sites are occupied by
contaminants, this leaves more sites to exclusively bind the target protein.
12. A pH of less than 6.5 can effect elution by protonating the histidine side chain,
preventing it from donating electrons to the bond with the IMCC.
13. A localized pH microenvironment may require more extreme shifts in pH to allow
elution.
14 . Alternative borderline Lewis metal ions will have different affinity for the histidine
tag. As a rule of thumb, binding strength is generally in the order Cu 2+ >Ni 2+ >
Co 2+ ≈ Zn 2+ (26), so the use of, for example, Zn 2+ may allow elution where it
was not possible from Ni 2+ .
15. Incorporation of the altered conditions into the binding and washing phase of the
chromatography run. It is often more effective to prevent non-specific interactions
from occurring that to disrupt them once established. In these circumstances, it
may be possible to achieve elution in the absence of the altered condition, as the
causative agent (or its effects) may remain loosely associated with the protein