No abstract

Surface functionalization of biological inert polymers (e.g., polypropylene PP; polystyrene PS) with material binding peptides facilitates an efficient immobilization of enzymes, bioactive peptides or antigens at ambient temperature in water. The developed robust directed evolution protocol enables to tailor polymer binding anchor peptides (PBPs) for efficient binding under application conditions. Key for a successful directed evolution campaign was to develop an epPCR protocol with a very high mutation frequency (60 mutations/kb) to ensure sufficient diversity in PBPs (47 aas LCI: “liquid chromatography peak I”; 44 aas TA2: “Tachystatin A2”). LCI and TA2 were genetically fused to the reporter egfp to quantify peptide binding on PP and PS by fluorescence analysis. The Peptide‐Polymer evolution protocol (PePevo protocol) was validated in two directed evolution campaigns for two PBPs and polymers (LCI: PP; TA2: PS). Surfactants were used as selection pressure for improved PBP binders (non‐ionic surfactant Triton X‐100; 1 mM for LCI‐PP // anionic surfactant LAS; 0.5 mM for TA2‐PS). PePevo yielded an up to three fold improved PP‐binder (LCI‐M1‐PP: I24T, Y29H, E42 K and LCI‐M2‐PP: D31V, E42G) and an up to six fold stronger PS‐binder (TA2‐M1‐PS: R3S, L6P, V12 K, S15P, C29R, R30L, F33S, Y44H and TA2‐M2‐PS: F9C, C24S, G26D, S31G, C41S, Y44Q).

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.