Water-soluble polyisocyanopeptides (PICs) are a new class of synthetic polymers that mimic natural protein-based filaments. Their unique semiflexible properties combined with a length of several hundred nanometers have recently enabled a number of biomedical applications ranging from tissue engineering to cancer immunotherapy. One crucial step toward the further development of PICs for these applications is the efficient and controlled synthesis and purification of PIC–biomolecule conjugates. Considering the large size of PICs and the biomolecules to be conjugated, conjugation reactions do usually not proceed to completion due to steric effects. As a consequence, purification of the reaction mixture is necessary to separate the obtained bioconjugates from unreacted biomolecules. As a direct result of the semiflexible nature of PICs, standard polymer and protein purification methods based on molecular weight have not been successful. Here, we introduce a new affinity-based purification method utilizing biotin as an affinity tag. PICs decorated with a controlled and tunable density of biotin molecules (biotinPICs) were efficiently bound to and eluted from a monoavidin resin in buffered aqueous solution. Using these biotinPICs, two different protein conjugates were synthesized, one carrying the enzyme alkaline phosphatase (PhoA) and the other T-cell activating anti-CD3 antibodies. The resulting biotinPIC–protein conjugates were successfully obtained in high purity (>90%) and without any loss of protein activity. The high purity greatly simplifies the analysis of biotinPIC bioconjugates, such as the determination of the average number of biomolecules conjugated per biotinPIC chain. Most importantly, it allows for the direct and straightforward application of the obtained bioconjugates in the desired applications. The new method developed may further be adapted for the purification of other advanced bioconjugates that are difficult to obtain in high purity with the available standard methods.

中文翻译：

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基于亲和力的多异氰肽生物共轭物的纯化

水溶性聚异氰肽（PIC）是模仿天然蛋白质基细丝的一类新型合成聚合物。它们独特的半柔韧性与数百纳米的长度相结合，最近使从组织工程到癌症免疫疗法的许多生物医学应用成为可能。对于这些应用，进一步开发PIC的关键步骤之一是PIC生物分子偶联物的高效可控合成和纯化。考虑到PIC和待结合的生物分子的大尺寸，由于空间效应，结合反应通常不会完全进行。结果，必须纯化反应混合物以将获得的生物缀合物与未反应的生物分子分离。由于事先知情同意的半灵活性质，基于分子量的标准聚合物和蛋白质纯化方法尚未成功。在这里，我们介绍了一种新的基于亲和力的纯化方法，该方法利用生物素作为亲和标签。装饰有受控且可调密度的生物素分子的PIC（biotinPICs）在缓冲水溶液中有效结合到Monoavidin树脂上并从中洗脱。使用这些生物素PIC合成了两种不同的蛋白质偶联物，一种偶联物带有碱性磷酸酶（PhoA），另一种则具有激活T细胞的抗CD3抗体。所获得的生物素PIC蛋白偶联物可以高纯度（> 90％）成功获得，而蛋白活性没有任何损失。高纯度极大地简化了生物素PIC生物缀合物的分析，例如确定每个生物素PIC链中缀合的生物分子的平均数。最重要的是，它允许将所获得的生物缀合物直接和直接地应用于所需的应用中。开发的新方法可能进一步适用于纯化其他先进的生物缀合物，而这些生物缀合物难以通过现有的标准方法获得高纯度。