A Platform for Developing High-Affinity Selective Binding Proteins to Short and Flexible Peptide Motifs

SUMMARY

• Short and flexible peptide motifs such as GPCRs and histone tails are attractive targets for therapeutic development, but the generation of traditional antibodies against these targets remains a challenge because of unfavorable binding kinetics.
• The inventors developed a novel affinity reagent scaffold particularly applicable to binding short and flexible peptide motifs consisting of a first capture domain that facilitates specific protein-protein interaction between the probe and target and a second enhancer domain that enhances the affinity to the target. The two connected domains work synergistically to achieve high levels of affinity and specificity that are unattainable with either domain alone. 
• The invention is a class of binding proteins called affinity clamps, with a clamshell architecture particularly applicable for binding short peptide motifs. Directed evolution with recombinant affinity clamp libraries can produce high affinity and specific binding reagents to a wide range of targets.
• In proof of concept experiments, affinity clamp libraries were used to explore the role of Grb2 interactions with specific phosphotyrosine motifs in PrE cell differentiation without relying on loss of function genetic knockdowns.  Affinity clamps were generated that could discriminate between similar phosphotyrosine peptide targets that bound with single digit nanomolar affinity.

FIGURE

(A)Schematic overview of the affinity clamp platform and screening process. Combinatorially diversified libraries are used to identify affinity clamps with a high affinity and specificity for a target of interest. (B) Protein ribbon structure of an affinity clamp that binds to a specific tag (yellow). The capture domain is shown in grey (PDZ) and the enhancer domain in green (FN3). 

ADVANTAGES

ADVANTAGES

• Works on challenging epitopes not compatible with traditional antibodies
• Highly expressible scaffold with low cost and scalable in vitro production
• Platform technology applicable to a wide range of different targets
• Applicable with directed evolution using both yeast and phage display

APPLICATIONS

• Antibody-based research assays
• Label free biosensors
• Affinity tag purification and pulldown assays  

PUBLICATIONS

• Koide S, Huang J. Generation of high-performance binding proteins for peptide motifs by affinity clamping. Methods Enzymol. 2013;523:285-302.
• Huang J, Nagy SS, Koide A, Rock RS, Koide S. A peptide tag system for facile purification and single-molecule immobilization. Biochemistry. 2009;48(50):11834-11836.
• Yasui N, Findlay GM, Gish GD, et al. Directed network wiring identifies a key protein interaction in embryonic stem cell differentiation. Mol Cell. 2014;54(6):1034-1041.

Jun 29, 2020

Licensing

Proof of concept in in vitro molecular biology assays and protein tag purification|Affinity clamps generated and characterized against various PDZ and PY epitopes

Patent Pending

Jin Huang

• Issued US Patents on affinity clamps and methods of use: 9,885,050 8,263,350
• Tangible property for material transfer (recombinant libraries)