Synthetic polymers are used in numerous applications (e.g. packaging, automobiles or furniture) and therefore play an essential role in our daily life. Surface functionalization is essential to combine required surface properties e.g. wettability, dyability, biocompatibility, or anti-biofouling, with polymer bulk properties. Standard surface modifications are based on photo-, plasma- or chemo-treatments. Surface modification based on peptide or protein adsorption on the other hand can reduce energy and water consumption of the process considerably and lead to fewer toxic waste. Material binding anchor peptides are ideal candidates for biological surface functionalization due to their propensity to selectively bind to surfaces. The knowledge about molecular modes of interactions between peptides, surfaces and solvents is until now limited. The directed evolution of binding peptides is challenging due to the limits in diversity generation in short peptides and in the screening of binding peptides. Knowledge gaining directed evolution approaches can provide first insights into peptides surface interactions while simultaneously tailoring the binding strength for application demands. Therefore, the aim of this work was to identify and characterize a polymer binding peptide and to provide first insight into its surface interactions in application conditions through the KnowVolution approach. A novel toolbox for identification of polymer binding anchor peptides was developed. Fusion proteins composed of anchor peptides (adenoregulin, catelicidin-BF, cecropin A, reutericin, and LCI) and enhanced green fluorescent protein (EGFP) were designed and their binding potential towards polypropylene surfaces was analyzed by fluorescence microscopy. LCI was identified as a high potential polypropylene binding peptide. Protein coatings of EGFP-LCI were characterized by fluorescence and scanning force microscopy. EGFP-LCI formed a densely packed monolayer of 4.1 ± 0.2 nm thickness and with a coating density of 0.8 pmol/cm2. The applicability of the anchor peptide toolbox for polypropylene functionalization was verified by equipping polypropylene with the fluorescent dye ThioGlo-1 via the anchor peptide LCI. Application of anchor peptides as adhesion promoters requires stable binding under process conditions (e.g. presence of surfactants). A robust directed evolution protocol (PePevo) was developed to tailor polymer binding peptides to specific application conditions. Keys for success were the development of an epPCR protocol with an extremely high mutation frequency (60 mutations/kb) to ensure sufficient mutations in polymer binding peptides and the optimization of screening assay (e.g. protein concentration and surfactant concentration) to achieve a standard deviation of ±14.4% (selection pressure 1 mM Triton X 100). Interactions between material surfaces, peptides, and solvent are often not sufficiently understood to enable a rational polymer binding peptide design. By employing the KnowVolution (knowledge gaining directed evolution) protein properties can be adapted to application demands. One round of KnowVolution was performed to gain insights into interactions between LCI and polypropylene in presence of the surfactant Triton X-100. KnowVolution yielded 8 LCI positions (D19, S27, Y29, D31, G35, I40, E42, and D45) which influence the binding to PP in presence of Triton X-100. Amino acid substitutions Y29R and G35R were recombined in variant LCI KR-2 with a 5.4 ± 0.5-fold stronger PP-binding in presence of Triton X-100 (1 mM). The variant LCI KR-2 shows a maximal binding capacity of 8.8 ± 0.1 pmol/cm2 on PP with a Triton X-100 up to 1 mM. The variant LCI KR-2 furthermore yields a 5.1-fold increased maximal binding capacity and a 1.9-fold increased dissociation constant. Interactions of anchor peptide LCI, PP, and Triton X-100 were shown to be affected by LCI’s negatively charged amino acid content. Variants with substituted negatively charged amino acids (D19, D31, E42, and D45) or variants with introduced positively charged amino acids bound stronger to PP than wild type LCI. Furthermore, it was noted that Triton X-100 influences the binding of LCI KR-2 very likely in its monomer form since an increase in concentration above critical micelle concentration (CMC: 0.2-0.9 mM) up to 10 mM did not influence the binding further. EGFP-LCI’s high coating density, the number and diversity of provided functional groups, and the adaptability to process conditions offer a feasible alternative to conventional modification strategies
