Lefevre Mathilde, Flammang Patrick, Aranko A Sesilja, Linder Markus B, Scheibel Thomas, Humenik Martin, Leclercq Maxime, Surin Mathieu, Tafforeau Lionel, Wattiez Ruddy, Leclère Philippe, Hennebert Elise
Laboratory of Cell Biology, Research Institute for Biosciences, University of Mons, Place du Parc 23, 7000 Mons, Belgium; Laboratory for Chemistry of Novel Materials, Research Institute for Materials, Center for Innovation and Research in Materials and Polymers (CIRMAP), University of Mons, Place du Parc 23, 7000 Mons, Belgium.
Biology of Marine Organisms and Biomimetics Unit, Research Institute for Biosciences, University of Mons, Place du Parc 23, 7000 Mons, Belgium.
Acta Biomater. 2020 Aug;112:62-74. doi: 10.1016/j.actbio.2020.05.036. Epub 2020 Jun 2.
Sea stars adhere to various underwater substrata using an efficient protein-based adhesive secretion. The protein Sfp1 is a major component of this secretion. In the natural glue, it is cleaved into four subunits (Sfp1 Alpha, Beta, Delta and Gamma) displaying specific domains which mediate protein-protein or protein-carbohydrate interactions. In this study, we used the bacterium E. coli to produce recombinantly two fragments of Sfp1 comprising most of its functional domains: the C-terminal part of the Beta subunit (rSfp1 Beta C-term) and the Delta subunit (rSfp1 Delta). Using native polyacrylamide gel electrophoresis and size exclusion chromatography, we show that the proteins self-assemble and form oligomers and aggregates in the presence of NaCl. Moreover, they adsorb onto glass and polystyrene upon addition of Na and/or Ca ions, forming homogeneous coatings or irregular meshworks, depending on the cation species and concentration. We show that coatings made of each of the two proteins have no cytotoxic effects on HeLa cells and even increase their proliferation. We propose that the Sfp1 recombinant protein coatings are valuable new materials with potential for cell culture or biomedical applications. STATEMENT OF SIGNIFICANCE: Biological adhesives offer impressive performance in their natural context and, therewith, the potential to inspire the development of advanced biomaterials for an increasing variety of applications in medicine or in material sciences. To date, most marine adhesive proteins that have been produced recombinantly in order to develop bio-inspired adhesives are small proteins from mussels and barnacles. Here, we produced two multi-modular proteins based on the sequence of Sfp1, a major protein from sea star adhesive secretion. These two proteins comprise most of Sfp1 functional domains which mediate protein-protein and protein-carbohydrate interactions. We characterized the two recombinant proteins with an emphasis on functional characteristics such as self-assembly, adsorption and cytocompatibility. We discuss their potential as biomaterials.
海星利用一种高效的基于蛋白质的粘性分泌物附着在各种水下基质上。蛋白质Sfp1是这种分泌物的主要成分。在天然胶水中，它被切割成四个亚基（Sfp1α、β、δ和γ），这些亚基具有特定的结构域，介导蛋白质-蛋白质或蛋白质-碳水化合物相互作用。在本研究中，我们利用大肠杆菌重组生产了Sfp1的两个片段，它们包含了其大部分功能结构域：β亚基的C末端部分（rSfp1βC-term）和δ亚基（rSfp1δ）。使用天然聚丙烯酰胺凝胶电泳和尺寸排阻色谱法，我们发现这些蛋白质在NaCl存在的情况下会自我组装并形成寡聚体和聚集体。此外，在添加Na和/或Ca离子后，它们会吸附在玻璃和聚苯乙烯上，根据阳离子种类和浓度形成均匀的涂层或不规则的网状结构。我们发现由这两种蛋白质中的每一种制成的涂层对HeLa细胞没有细胞毒性作用，甚至还能促进其增殖。我们提出，Sfp1重组蛋白涂层是有价值的新材料，具有用于细胞培养或生物医学应用的潜力。重要性声明：生物粘合剂在其天然环境中表现出令人印象深刻的性能，因此有潜力激发先进生物材料的开发，以用于医学或材料科学中越来越多的应用。迄今为止，为了开发受生物启发的粘合剂而重组生产的大多数海洋粘附蛋白都是来自贻贝和藤壶的小蛋白。在这里，我们基于海星粘性分泌物中的主要蛋白质Sfp1的序列生产了两种多模块蛋白质。这两种蛋白质包含了Sfp1的大部分功能结构域，这些结构域介导蛋白质-蛋白质和蛋白质-碳水化合物相互作用。我们对这两种重组蛋白进行了表征，重点关注其自组装、吸附和细胞相容性等功能特性。我们讨论了它们作为生物材料的潜力。
