Directed evolution of polypropylene and polystyrene binding peptides.

Area of Science:

• Biomaterials Science
• Protein Engineering
• Polymer Chemistry

Background:

• Biological inert polymers like polypropylene (PP) and polystyrene (PS) require surface functionalization for biomolecule immobilization.
• Material binding peptides (PBPs) offer a route to anchor enzymes, peptides, or antigens onto polymer surfaces at ambient temperatures.
• Directed evolution is a powerful tool for tailoring peptide binders for specific polymer applications.

Purpose of the Study:

• To develop and validate a robust directed evolution protocol (PePevo) for creating highly effective polymer binding anchor peptides (PBPs).
• To enhance the binding affinity of peptides to polypropylene and polystyrene surfaces for improved biomolecule immobilization.
• To demonstrate the efficacy of the PePevo protocol using model peptides and polymers under selection pressure.

Main Methods:

• Employed error-prone PCR (epPCR) with high mutation frequency (60 mutations/kb) to generate diverse PBP libraries.
• Fused peptides (LCI, TA2) to a reporter gene (egfp) for quantifying binding via fluorescence analysis on PP and PS.
• Utilized surfactant-based selection pressure (Triton X-100 for LCI-PP, LAS for TA2-PS) within the PePevo protocol.

Main Results:

• Developed tailored PBPs (LCI-M1/M2 for PP, TA2-M1/M2 for PS) with significantly enhanced binding affinities.
• Achieved up to a three-fold improvement in PP-binding for LCI variants.
• Demonstrated up to a six-fold increase in PS-binding for TA2 variants.

Conclusions:

• The PePevo protocol is a validated and effective method for generating high-affinity polymer binding peptides.
• This approach enables efficient surface functionalization of inert polymers for applications in biomolecule immobilization.
• The tailored PBPs show promise for advanced biomaterial development and biotechnological applications.