KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

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.

Matter‐tag: A universal immobilization platform for enzymes on polymers, metals, and silicon‐based materials

“…The dissociation constant ( K D ) of YmPh‐LCI was determined to be 2.9·10 −8 M and is comparable to the reported K D values of anti‐His‐tag antibodies (3D5 antibody: 3.4·10 −7 M, PentaHis antibody: 1.0·10 −9 M; Müller, Arndt, Bauer, & Plückthun, ) and polyhistidine‐tags for Ni 2+ ‐NTA (free 6His‐peptide: 1.4·10 −8 M; Knecht, Ricklin, Eberle, & Ernst, ). The K D values can be further reduced to promote stronger adhesion of the Matter‐ tags by applying directed evolution methodologies for instance PeP evo protocol (Rübsam, Weber et al, ) within a KnowVolution campaign (Rübsam, Davari et al, ).…”

“…Here, we report a universal immobilization platform based on adhesion-promoting peptides (Matter-tags). Three adhesion promoting peptides, CecA, LCI, and TA2 were selected due to their reported binding to synthetic polymers (PS, PP, PUR, triblock copolymers like PIB 1000 -PEG 6000 -PIB 1000 and PMOXA 15 -PDMS 68 -PMOXA 15 ; Islam, Apitius, Jakob, & Schwaneberg, 2019;Klermund, Poschenrieder, & Castiglione, 2016;Noor et al, 2012;Rübsam et al, 2017;Rübsam, Weber et al, 2018;Rübsam, Davari, Jakob, & Schwaneberg, 2018), and natural surfaces, for example, plant leaves (Meurer et al, 2017;Schwinges et al, 2019). The developed immobilization platform enables to cover a broad spectrum of industrially and medically relevant materials.…”

Ultrahigh‐throughput screening system for directed polymer binding peptide evolution

“…The LCI‐CSD library was generated by simultaneous SSM of five LCI positions (Y29, D31, G35, E42, and D45). These five positions were reported in a KnowVolution campaign to be key contact positions for PP‐binding in the presence of the nonionic surfactant Triton X‐100 (Rübsam et al, ). The LCI‐CSD library was generated using degenerative primers with five NNK positions, resulting in up to 3.2 million peptide variants.…”

“…eGFP‐LCI WT: 45965 M −1 cm −1 , 37.1 kDa; eGFP‐LCI‐CSD‐3: 45840 M −1 cm −1 , 36.9 kDa. According to the determined protein concentrations, serial dilutions (0‐1 µM) of both variants were prepared and B Max values were determined as published by Rübsam et al ().…”

“…The peptide was furthermore optimized in a directed peptide evolution campaign (PeP evo , epPCR based) for strengthened PP‐binding in the presence of Triton X‐100 (Rubsam, Jakob, & Schwaneberg, ). Additionally, LCI was engineered in a knowledge gaining directed evolution (KnowVolution) campaign reporting a 5.4‐fold improved PP‐binder (Y29R and G35R) in the presence of Triton X‐100 (Rübsam, Jakob, & Schwaneberg, ). Both, the PeP evo and KnowVolution campaign were performed in MTP format by screening around 1,000 clones and requiring lysis of E. coli cells to determine improved binding strength.…”