RWTH Aachen University, Worringerweg 3, Aachen, Germany.
DWI - Leibniz-Institute for Interactive Materials, Forckenbeckstraße 50, Aachen, Germany.
Biotechnol Bioeng. 2018 Feb;115(2):321-330. doi: 10.1002/bit.26481. Epub 2017 Nov 15.
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).
生物惰性聚合物（如聚丙烯（PP）；聚苯乙烯（PS））的表面功能化与材料结合肽相结合，可在环境温度下将酶、生物活性肽或抗原高效固定在水中。开发的稳健定向进化方案可定制聚合物结合锚定肽（PBPs），以在应用条件下实现高效结合。成功进行定向进化运动的关键是开发一种具有非常高突变频率（60 个突变/kb）的 epPCR 方案，以确保 PBPs 具有足够的多样性（47 个氨基酸 LCI：“液相色谱峰 I”；44 个氨基酸 TA2：“Tachystatin A2”）。LCI 和 TA2 被遗传融合到报告基因 egfp 中，通过荧光分析定量 PP 和 PS 上的肽结合。肽-聚合物进化方案（PePevo 方案）已在两个定向进化运动中得到验证，用于两种 PBPs 和聚合物（LCI：PP；TA2：PS）。表面活性剂被用作改进 PBP 结合物的选择压力（非离子表面活性剂 Triton X-100；LCI-PP 的 1mM//阴离子表面活性剂 LAS；TA2-PS 的 0.5mM）。PePevo 产生了高达三倍的改进的 PP 结合物（LCI-M1-PP：I24T、Y29H、E42K 和 LCI-M2-PP：D31V、E42G）和高达六倍的更强的 PS 结合物（TA2-M1-PS：R3S、L6P、V12K、S15P、C29R、R30L、F33S、Y44H 和 TA2-M2-PS：F9C、C24S、G26D、S31G、C41S、Y44Q）。
