Biology Reference
In-Depth Information
a column containing immobilized nickel, only the His-tag protein binds and all other proteins
can be discarded. The His-tag protein can be eluted from the column by an imidazole-
containing solution. Imidazole, the side chain of histidine, competes for the poly-histidine
binding site. The His-tag protein can also be released from the nickel column by lowering
the pH to ~4.5. It should be mentioned that any protein with a high affinity for divalent
metals can be separated by an immobilized-nickel column without the need of a special
His-tag.
Another approach to purification of a protein through use of an engineered tag involves
addition of an antigen peptide tag. Separation is achieved using an appropriate antibody-
attached resin column. This methodology is highly specific to a single protein. After the
desired protein is isolated, the engineered tags can be easily removed by a protease.
After the desired protein is obtained in pure form, it is usually so dilute it must be sub-
stantially concentrated for subsequent reconstitution studies. The most commonly used
concentration technique is lyphilization or freeze-drying. The sample is frozen and the
water content reduced by sublimation under vacuum. Another procedure increases the
protein concentration by ultrafiltration. Selectively permeable membranes allow water
and small molecules to cross the membrane while retaining the much larger protein. The
solution is forced through the filter by mechanical or gas pressure or by centrifugal force
in a specially designed centrifuge tube.
At each step in the multi-step protein purification process, both the total amount of all
proteins present and the activity of the desired protein must be determined. Although the
total protein and total activity of the desired protein will decrease after each step, the specific
activity will increase if any purification is achieved (
Figure 13.4
). Enzyme activity is equal to
the moles of substrate converted to product per unit of time. The standard unit of activity is
the katal where:
1
katal
¼ 1
mol converted
=
sec
However a katal is such a large unit, specific activity is more usually expressed as:
enzyme unit
U
¼ 110
6
mol substrate converted
1
=
min
Therefore, 1 U corresponds to 16.67 x 10
9
katals.
Specific
Activity
Total
Activity
1
2
3
4
5
PURIFICATION STEPS
FIGURE 13.4
With each step in the purification process, the total enzyme activity decreases while specific
activity increases until the enzyme is pure.
