PDF(Pubmed)
Abstract：
Protein conjugation to biomaterial scaffolds is a powerful approach for tissue engineering. However, typical chemical conjugation methods lack site-selectivity and can negatively impact protein bioactivity. To overcome this problem, a site-selective strategy is reported here for installing tetrazine groups on terminal poly-histidines (His-tags) of recombinant proteins. These tetrazine groups are then leveraged for bio-orthogonal conjugation to poly(ethylene glycol) (PEG) hydrogel microparticles, which are subsequently assembled into microporous annealed particle (MAP) hydrogels. Efficacy of the strategy is demonstrated using recombinant, green fluorescent protein with a His tag (His-GFP), which enhanced fluorescence of the MAP hydrogels compared to control protein lacking tetrazine groups. Subsequently, to demonstrate efficacy with a therapeutic protein, recombinant human bone morphogenetic protein-2 (His-BMP2) was conjugated. Human mesenchymal stem cells growing in the MAP hydrogels responded to the conjugated BMP2 and significantly increased mineralization after 21 days compared to controls. Thus, this site-selective protein modification strategy coupled with bio-orthogonal click chemistry is expected to be useful for bone defect repair and regeneration therapies. Broader application to the integration of protein therapeutics with biomaterials is also envisioned.
摘要：
蛋白质与生物材料支架的结合是组织工程的有效方法。然而,典型的化学缀合方法缺乏位点选择性，并且会对蛋白质的生物活性产生负面影响。为了克服这个问题，本文报道了一种位点选择性策略，用于在重组蛋白的末端聚组氨酸（His标签）上安装四嗪基团。然后利用这些四嗪基团与聚（乙二醇）（PEG）水凝胶微粒进行生物正交缀合，其随后组装成微孔退火颗粒(MAP)水凝胶。使用重组体证明了该策略的功效，带有His标记的绿色荧光蛋白（His-GFP），与缺乏四嗪基团的对照蛋白相比，MAP水凝胶的荧光增强。随后，为了证明治疗性蛋白质的功效，重组人骨形态发生蛋白-2(His-BMP2)缀合。与对照相比，在MAP水凝胶中生长的人间充质干细胞对缀合的BMP2有反应，并在21天后显着增加了矿化。因此，这种位点选择性蛋白修饰策略结合生物正交点击化学有望用于骨缺损修复和再生治疗.还设想了将蛋白质治疗剂与生物材料整合的更广泛的应用。
