期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 115, 期 2, 页码 321-330出版社
WILEY
DOI: 10.1002/bit.26481
关键词
anchor peptides; directed evolution; polymer binding peptides; polystyrene; polypropylene; surface functionalization
资金
- Allianz Industrie ForschungIGF-Vorhaben [18180 N]
- Bundesministerium fur Bildung und Forschung [FKZ: 031A227F]
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; 1mM for LCI-PP // anionic surfactant LAS; 0.5mM for TA2-PS). PePevo yielded an up to three fold improved PP-binder (LCI-M1-PP: I24T, Y29H, E42K and LCI-M2-PP: D31V, E42G) and an up to six fold stronger PS-binder (TA2-M1-PS: R3S, L6P, V12K, S15P, C29R, R30L, F33S, Y44H and TA2-M2-PS: F9C, C24S, G26D, S31G, C41S, Y44Q).
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