Random Mutagenesis of Peptide Aptamers as an Optimization Strategy for Inhibitor Screening
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Accumulating work over the past decade has shown that peptide aptamer screening represents a valid strategy for inhibitor identification that can be applied to a variety of different targets. Because of the screening method in cells and the highly combinatorial libraries available, this approach yields rapidly highly specific candidate inhibitors. Once a hit peptide has been identified, its interaction strength and affinity towards its target protein can be optimized even more, in order to increase its inhibition efficiency when subsequently applied in vivo. A condition to a successful optimization is that gain of inhibition strength should not result in loss of specificity.
Here we present a simple method for peptide aptamer optimization, which can be achieved by PCR-based random mutagenesis combined with a selection screen in yeast using a strong selective drug. The rationale of this approach, which has proven valid and efficient, is that stronger interaction in yeast will also lead to stronger inhibition. Our optimization method is effective, without loss of specificity, which is of a great importance for the discovery of inhibitors that target specific protein–protein interactions.
Key wordsPeptide aptamers PCR-based mutagenesis Yeast two-hybrid Fluorescent GEF assay Inhibitor screening
We are grateful to Anne Briançon-Marjollet for her contribution to the library construction. This work was supported by grants from the ANR “Physique et chimie du vivant” grant N° 06-137373. N. B. was supported by a CNRS valorization fellowship from CNRS. All authors are members of the CNRS consortium GDR2823.
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