López Barreiro Diego, Houben Klaartje, Schouten Olaf, Koenderink Gijsje H, Thies Jens C, Sagt Cees M J
Manufacturing Futures Lab, Department of Chemical Engineering, University College London, London WC1E 7JE, United Kingdom.
Centre for Nature-Inspired Engineering, Department of Chemical Engineering, University College London, London WC1E 7JE, United Kingdom.
ACS Appl Mater Interfaces. 2025 Jan 8;17(1):650-662. doi: 10.1021/acsami.4c17903. Epub 2024 Dec 16.
The biofabrication of recombinant structural proteins with a range of mechanical or structural features usually relies on the generation of protein libraries displaying variations in terms of amino acid composition, block structure, molecular weight, or physical/chemical cross-linking sites. This approach, while highly successful in generating a wealth of knowledge regarding the links between design features and material properties, has some inherent limitations related to its low throughput. This slows down the pace of the development of recombinant structural proteins. Here, we propose an approach to tune the viscoelastic properties of temperature-responsive hydrogels made of silk-elastin-like polypeptides (SELPs) without modifying their sequence. To do so, we subject purified SELPs to two different postprocessing methods─water annealing or EtOH annealing─that alter the topology of highly disordered SELP networks via the formation of ordered intermolecular β-sheet physical cross-links. Combining different analytical techniques, we connect the order/disorder balance in SELPs with their gelling behavior. Furthermore, we show that introducing a functional block (in this case, a biomineralizing peptide) in the sequence of SELPs can disrupt its self-assembly and that such disruption can only be overcome by EtOH annealing. Our results suggest that postprocessing of as-purified SELPs might be a simple approach to tune the self-assembly of SELPs into biomaterials with bespoke viscoelastic properties beyond the traditional approach of developing SELP libraries via genetic engineering.
具有一系列机械或结构特征的重组结构蛋白的生物制造通常依赖于生成蛋白质文库，这些文库在氨基酸组成、嵌段结构、分子量或物理/化学交联位点方面存在差异。这种方法虽然在生成大量有关设计特征与材料特性之间联系的知识方面非常成功，但由于其低通量存在一些固有局限性。这减缓了重组结构蛋白的开发速度。在此，我们提出一种方法，在不改变其序列的情况下调节由丝弹性蛋白样多肽（SELP）制成的温度响应水凝胶的粘弹性特性。为此，我们对纯化的SELP进行两种不同的后处理方法——水退火或乙醇退火——通过形成有序的分子间β-折叠物理交联来改变高度无序的SELP网络的拓扑结构。结合不同的分析技术，我们将SELP中的有序/无序平衡与其凝胶化行为联系起来。此外，我们表明在SELP序列中引入功能嵌段（在这种情况下，一种生物矿化肽）会破坏其自组装，并且这种破坏只能通过乙醇退火来克服。我们的结果表明，纯化后的SELP的后处理可能是一种简单的方法，可将SELP的自组装调节为具有定制粘弹性特性的生物材料，这超出了通过基因工程开发SELP文库的传统方法。
