Peters Daniel T, Waller Helen, Birch Mark A, Lakey Jeremy H
1Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, UK.
2Division of Trauma and Orthopaedic Surgery, Department of Surgery, University of Cambridge, Cambridge, UK.
J Biol Eng. 2019 Jun 18;13:54. doi: 10.1186/s13036-019-0183-2. eCollection 2019.
Engineered living materials (ELMs) are an exciting new frontier, where living organisms create highly functional materials. In particular, protein ELMs have the advantage that their properties can be manipulated via simple molecular biology. Caf1 is a protein ELM that is especially attractive as a biomaterial on account of its unique combination of properties: bacterial cells export it as a massive, modular, non-covalent polymer which is resistant to thermal and chemical degradation and free from animal material. Moreover, it is biologically inert, allowing the bioactivity of each 15 kDa monomeric Caf1 subunit to be specifically engineered by mutagenesis and co-expressed in the same cell to produce a mixture of bioactive Caf1 subunits.
Here, we show by gel electrophoresis and transmission electron microscopy that the bacterial cells combine these subunits into true mosaic heteropolymers. By combining two separate bioactive motifs in a single mosaic polymer we demonstrate its utility by stimulating the early stages of bone formation by primary human bone marrow stromal cells. Finally, using a synthetic biology approach, we engineer a mosaic of three components, demonstrating that Caf1 complexity depends solely upon the variety of monomers available.
These results demonstrate the utility of engineered Caf1 mosaic polymers as a simple route towards the production of multifunctional biomaterials that will be useful in biomedical applications such as 3D tissue culture and wound healing. Additionally, in situ Caf1 producing cells could create complex bacterial communities for biotechnology.
工程化活材料（ELMs）是一个令人兴奋的新领域，在这个领域中，活生物体能够制造出具有高度功能的材料。特别是，蛋白质ELMs具有这样的优势，即其特性可以通过简单的分子生物学方法进行调控。Caf1是一种蛋白质ELM，由于其独特的特性组合，作为一种生物材料特别具有吸引力：细菌细胞将其作为一种大量的、模块化的非共价聚合物分泌出来，这种聚合物耐热且耐化学降解，并且不含动物材料。此外，它具有生物惰性，这使得每个15 kDa的单体Caf1亚基的生物活性能够通过诱变进行特异性改造，并在同一细胞中共表达，以产生具有生物活性的Caf1亚基混合物。
在这里，我们通过凝胶电泳和透射电子显微镜表明，细菌细胞将这些亚基组合成真正的镶嵌杂聚物。通过在单个镶嵌聚合物中组合两个单独的生物活性基序，我们通过刺激原代人骨髓基质细胞的骨形成早期阶段证明了其效用。最后，使用合成生物学方法，我们构建了一个由三个组分组成的镶嵌体，证明了Caf1的复杂性仅取决于可用单体的种类。
这些结果证明了工程化Caf1镶嵌聚合物作为生产多功能生物材料的简单途径的效用，这些生物材料将在诸如3D组织培养和伤口愈合等生物医学应用中发挥作用。此外，原位产生Caf1的细胞可以为生物技术创造复杂的细菌群落。
