Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries

Area of Science:

• Molecular Biology
• Protein Engineering
• Biochemistry

Background:

• MAP-kinases play crucial roles in cellular signaling pathways.
• Developing specific inhibitors for MAP-kinases is vital for therapeutic applications.
• Designed ankyrin repeat proteins (DARPins) offer a versatile scaffold for protein engineering.

Purpose of the Study:

• To rapidly select specific MAP-kinase binders using a combinatorial library of DARPins.
• To evaluate the efficiency of a combined ribosome display and protein fragment complementation assay (PCA) selection strategy.
• To generate DARPins functional within the cellular cytoplasm.

Main Methods:

• Utilized ribosome display for in vitro selection of DARPins.
• Employed protein fragment complementation assay (PCA) for in vivo selection and validation.
• Applied a combined in vitro/in vivo approach for rapid binder enrichment.
• Tested DARPins against JNK1, JNK2, and p38 kinases.

Main Results:

• Isolated specific JNK2-binding DARPins with micromolar affinity after one round of selection.
• Achieved low nanomolar affinities for JNK2-specific DARPins after two rounds of selection.
• Demonstrated high specificity of identified DARPins for target kinases (JNK1, JNK2, p38).
• Observed enrichment factors approaching 10^5 per round of ribosome display.

Conclusions:

• The combination of ribosome display and PCA enables rapid identification of specific protein binders.
• DARPins selected through this method are functional in the cellular cytoplasm.
• This approach significantly accelerates the discovery of targeted protein binders for kinases.