Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
DWI - Leibniz Institute for Interactive Materials, Aachen, Germany.
Biotechnol Bioeng. 2021 Apr;118(4):1520-1530. doi: 10.1002/bit.27666. Epub 2021 Feb 4.
A versatile peptide-based toolbox for surface functionalization was established by a combination of a universal material binding peptide (LCI-anchor peptide) and sortase-mediated bioconjugation (sortagging). This toolbox facilitates surface functionalization either as a one- or a two-step strategy. In the case of the one-step strategy, the desired functionality was directly introduced to LCI. For the two-step strategy, LCI was modified with a reactive group, which can be further functionalized (e.g., employing "click" chemistry). Sortagging of LCI, employing sortase A from Staphylococcus aureus, was achieved with six different amine compounds: dibenzocyclooctyne amine, biotin-polyethylene glycol amine, Cyanine-3 amine, kanamycin, methoxypolyethylene glycol amine (Mn = 5000 Da), and 2,2,3,3,4,4,4-Heptafluorobutylamine. The purification of LCI-amine sortagging products was performed by a negative purification using Strep-tag II affinity chromatography, resulting in LCI-amine conjugates with purities >90%. For the two-step strategy, the LCI-dibenzocyclooctyne sortagging product was purified and enabled, through copper-free azide-alkyne "click" chemistry, universal surface functionalization of material surfaces such as polypropylene, polyethylene terephthalate, stainless steel, gold, and silicon. The click reaction was performed before or after surface binding of LCI-dibenzocyclooctyne. Finally, in the case of the one-step strategy, polypropylene was directly functionalized with Cyanine-3 and biotin-polyethylene glycol amine.
建立了一个通用的材料结合肽(LCI-锚定肽)和 sortase 介导的生物偶联(sortagging)相结合的多功能肽基工具箱,用于表面功能化。该工具箱可通过一步或两步策略实现表面功能化。在一步策略中,直接将所需的功能引入 LCI。对于两步策略,LCI 用反应性基团进行修饰,该基团可以进一步进行功能化(例如,采用“点击”化学)。使用金黄色葡萄球菌的 sortase A 对 LCI 进行 sortagging,采用了六种不同的胺化合物:二苯并环辛炔胺、生物素-聚乙二醇胺、Cy3 胺、卡那霉素、甲氧基聚乙二醇胺(Mn = 5000 Da)和 2,2,3,3,4,4,4-七氟丁基胺。使用 Strep-tag II 亲和层析通过负纯化对 LCI-胺 sortagging 产物进行纯化,得到纯度>90%的 LCI-胺缀合物。对于两步策略,对 LCI-二苯并环辛炔 sortagging 产物进行纯化,并通过无铜叠氮-炔基“点击”化学实现了对聚丙稀、聚对苯二甲酸乙二酯、不锈钢、金和硅等材料表面的通用表面功能化。在 LCI-二苯并环辛炔表面结合之前或之后进行点击反应。最后,在一步策略中,直接将 Cy3 和生物素-聚乙二醇胺与聚丙稀进行功能化。