Selection of a cyclic nonapeptide inhibitor to α-chymotrypsin using a phage display peptide library |
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Authors: | Krook Margareta Lindbladh Christer Eriksen Jon Amund Mosbach Klaus |
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Institution: | (1) Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S–221 00 Lund, Sweden |
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Abstract: | A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against α-chymotrypsin (EC 3.4.21.1)
at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions,
interacted more strongly with α-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected
phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions.
The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit
the enzymatic activity of α-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only
marginally inhibited by the same peptide under the same conditions. The inhibition constant for α-chymotrypsin was estimated
to be 10-6 M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified α-chymotrypsin than
for natural α-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively
prevented from binding to α-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest
that the oxidized form of the selected peptide Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of α-chymotrypsin
and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys
has not been found in nature.
This revised version was published online in August 2006 with corrections to the Cover Date. |
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Keywords: | α -chymotrypsin combinatorial library cyclic peptide inhibitor phage display |
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