Engineered adhesion peptides for functionalization of natural surfaces, polymers, and metal alloys

  • Maßgeschneiderte Adhäsionspeptide für die Funktionalisierung von natürlichen Oberflächen, Polymeren und Metalllegierungen

Apitius, Lina; Schwaneberg, Ulrich (Thesis advisor); Pich, Andrij (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

Plastics or synthetic organic polymers have been produced in large scale since the 1950´s. Until today, roughly 8300 million metric tons of virgin plastics have been produced for an uncountable number of applications. Despite favorable bulk properties, the surface characteristics of polymers are often not ideal for intended applications (e.g., wettability, anti-static, anti-fouling, and bio-compatibility) and are mainly engineered by physical or chemical means. UV-radiation, flame/plasma or wet-chemical treatments often generate large volumes of toxic waste or safety hazard. Anchor peptides enable a biological surface functionalization at very high densities (monolayers) of peptides. By the use of anchor peptides multiple functionalities (e.g., NH2 , COOH- or OH-groups) can be introduced simultaneously. Anchor peptides are highly diverse in length, secondary structure, binding affinity as well as binding strength. In order to identify suitable target binder, a general anchor peptide platform was established using the fluorescent eGFP as reporter protein in binding studies. Identified binders need to perform under application conditions, therefore, anchor peptides have to be adapted by directed peptide evolution. Anchor peptides are very short in length (12-100 amino acids) and conventional diversity generation is challenging due to a low mutational rate of existing methods. To overcome this drawback, a Peptide-Polymer evolution protocol (PePevo protocol) was established. The anchor peptide Tachystatin A2 was randomized by epPCR employing a very high mutation frequency (59 mutations/kb) and was evolved for improved polystyrene binding strength. The fusion partner eGFP was used to quantify peptide binding in a micro-titer plate-based fluorescent assay. Therefore, the key performance parameters protein concentration, standard deviation and selection pressure were optimized. The anionic surfactant sodium dodecylbenzenesulfonate (LAS; 0.5 mM) was applied as selection pressure to identify improved polystyrene-binding Tachystatin A2 variants. Finally, the PePevo protocol yielded an up to six-fold stronger polystyrene-binder in the presence of 0.5 mM LAS in one round of directed evolution. One bottleneck of directed evolution is the screening throughput that is needed to analyze a meaningful amount of generated peptide diversity. An E. coli cell surface display technology for the engineering of anchor peptides was development, that enabled screening of large libraries (>10$^{6}$). The polypropylene-binding peptide LCI was fused as a passenger peptide to the cell-surface presented esterase A. LCI immobilized the E. coli cells on polypropylene beads and improved binding variants were enriched from culture broth. In a semi-rational approach, five LCI positions were saturated simultaneously. One round of directed evolution yielded an up to twelve-fold stronger polypropylene binder in the presence of 0.125 mM LAS. The usage of polymers for medical applications (e.g., stents or catheters) is often hampered by a low bio-compatibility. Medical implants often cause severe infections in patients and are therefore often coated with drug- or antibiotic-releasing systems. The versatility of anchor peptides facilitates the generation of biadhesive peptides that bind chemically and structurally different surfaces whereby the biadhesive peptide functions as a glue. A biadhesive peptide was generated that was composed of one stainless steel binding anchor peptide and one polycaprolactone binding anchor peptide. The generated biadhesive peptide Dermaseptin S1-Domain Z-LCI immobilized antibiotic-loaded polycaprolactone micro-containers on stainless steel surfaces, enabling the construction of drug eluting bare-metal stents. The antimicrobial activity of kanamycin-releasing polycaprolactone coated stainless steel wires was confirmed by growth inhibition of E. coli cells.Microplastics lingering in the environment are of increasing concern for human health. Enzymatic degradation of microplastic particles such as polyurethane is a promising strategy to convert plastic waste into carbon dioxide, monomers, and valuable compounds. A fusion protein composed of the polyester-polyurethane degrading cutinase Tcur1278 and the anchor peptide Tachystatin A2 was generated. Tachystatin A2 directed the enzyme (Tcur1278) to polyester-polyurethane nanoparticles. The anchor peptide enhanced the degradation kinetics and reduced the degradation half-life of the polymer significantly (both up to six-fold) in highly diluted suspensions at ambient temperature. The utilization of tunable anchor peptides enables a targeted microplastic degradation e.g., in wastewater treatment plants.

Identifier

Downloads