Abstract
The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents.
Highlights
Protein-based adhesion promoters enable directed protein and peptide immobilization onto material surfaces with a broad range of applications in enzyme catalysis, anti-microbial coatings, drug delivery systems, and biosensors [1,2,3,4]
Phase IV consists of the recombination of beneficial amino acid substitutions, which resulted in the generation of the variant EGFP-LCI KR-2
The second part describes the characterization of the identified LCI variants in comparison to the wild type with respect to binding strengths on polypropylene in the presence of Triton X-100
Summary
Protein-based adhesion promoters enable directed protein and peptide immobilization onto material surfaces with a broad range of applications in enzyme catalysis, anti-microbial coatings, drug delivery systems, and biosensors [1,2,3,4]. PBPs were found to recognize and bind to polymer surfaces via π–π interactions, hydrophobic and electrostatic interactions, and hydrogen bonding [7,8,9,10]. PBP binding depends on the peptide conformation, the amino acid content and the chemical composition, the molecular structure, the size, and the shape of the polymer [11]. The binding mechanism of these aromatic amino acids and the aromatic moieties of polystyrene is primarily based upon the stacking of aromatic ring systems [13,14]. Poly(methyl methacrylate) (PMMA)-binding peptide c2 was found to rely on the C-terminal proline residue resulting in a conformation capable of forming hydrogen bonding to ester groups in PMMA [14]
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