Affinity Chromatography - Department of Molecular and Cellular ...

Purification or removal of serine proteases, e.g. thrombin and
trypsin, and zymogens
HiTrap Benzamidine FF (high sub), Benzamidine Sepharose 4 Fast Flow (high sub)
Sample extraction procedures often release proteases into solution, requiring the addition
of protease inhibitors to prevent unwanted proteolysis. An alternative to the addition of
inhibitors is to use a group specific affinity medium to remove the proteases from the
sample. The same procedure can be used to either specifically remove these proteases or
purify them.
The synthetic inhibitor para-aminobenzamidine is used as the affinity ligand for trypsin,
trypsin-like serine proteases and zymogens. Benzamidine Sepharose 4 Fast Flow (high sub)
is frequently used to remove molecules from cell culture supernatant, bacterial lysate or
serum. During the production of recombinant proteins, tags such as GST are often used to
facilitate purification and detection. Enzyme specific recognition sites are included in the
recombinant protein to allow the removal of the tag by enzymatic cleavage when required.
Thrombin is commonly used for enzymatic cleavage, and must often be removed from the
recombinant product. HiTrap Benzamidine FF (high sub) provides a simple, ready to use
solution for this process. Figure 29 shows the partial structure of Benzamidine Sepharose 4
Fast Flow (high sub) and Table 4 gives examples of different serine proteases.
S
epharose
O
OH OH H
O
O
N
N
H
NH
NH 2
Fig. 29. Partial structure of Benzamidine Sepharose 4 Fast Flow (high sub).
Table 4. Examples of different serine proteases.
Source M r pI
Thrombin Bovine pancreas 23 345 10.5
Trypsin Human plasma chain A 5 700
Human plasma chain B 31 000 7.1
Urokinase Human urine 54 000 8.9
Enterokinase Porcine intestine heavy chain 134 000 4.2
Porcine intestine light chain 62 000
Plasminogen Human plasma 90 000 6.4–8.5
Prekallikrein Human plasma nd nd
Kallikrein Human plasma 86 000 nd (plasma)
Human saliva nd 4.0 (saliva)
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