Lean bio-manufacture of surface-binding proteins for multi-functional coatings

重组蛋白在生物医学和生物技术方面有许多应用，其中一些要求它们在非生物条件下是稳定的。这导致了将蛋白质固定在表面的需求。然而，由于要求保持表面材料的性质，以及在整个生产、纯化和表面结合的整个过程中保持蛋白质的活性，可能会出现问题。此外，当生产的各个阶段分开时，可能会出现不可预见的错误。该项目建议使用巴斯德毕赤酵母模块克隆(MoClo)工具包以及酵母MoClo工具包，并结合统计实验设计(DoE)方法来生产快速、基于蛋白质的多功能材料的自动化系统。该项目有四个主要目标：i.开发巴斯德毕赤酵母作为表达宿主来生产表面结合蛋白；ii.验证巴斯德毕赤酵母用于生产与多功能涂料制造相关的表面结合蛋白的有效性；iii.解决标准生物设计实践中“一次优化一个因素”的问题。iv.扩大酶和表面材料的范围。这将包括使用金属纳米颗粒作为表面，因为已经证明它们直接被蛋白质覆盖，从而产生稳定的金属纳米颗粒，从而保持蛋白质的活性。这些目标和目标将通过以下程序实现。首先，利用MoClo酵母工具包，结合MoClo毕赤酵母工具包，构建含有不同启动子、分泌标签和荧光报告基因的盒式表达载体，转化大肠杆菌，然后通过电穿孔转化巴斯德毕赤酵母。由于在毕赤酵母中缺乏稳定的表达系统，质粒盒将通过同源重组的方式整合到基因组中，与基于质粒的表达相比，增加了稳定性。通过测量总荧光来评估细胞的分泌效率，然后通过离心去除细胞后测定上清液的荧光来评估细胞的分泌效率。来自iGEM特征纤维素结合域注册表的纤维素结合蛋白(CBP)将被融合到荧光报告上。它与纤维素结合的能力将随着纯度的增加而分析，例如与介质中的细胞结合，通过离心法、离子交换层析法和凝胶过滤层析法去除细胞。卓别林将接受测试，因为有物种，如网状链霉菌，含有编码参与纤维素结合的蛋白质的基因。由于C2-甲壳素结合蛋白与纤维素结合蛋白相似，并具有与纤维素结合的能力，因此将对该蛋白进行检测。采用DOE方法，所有相关生产、纯化和表面结合性质的参数将同时改变。能源部的好处是，通过独立识别和评估交互作用的效果，可以更有效地搜索优化。在建立了用于自动生产纤维素结合蛋白的原则性能源部模型之后，可以探索酶和表面材料的范围。金属-纳米粒子具有很高的比表面积与体积比以及等离子体或磁性，因此具有很高的表面结合效率和简单的检测和分离能力，因此值得研究。我们将生产金属结合蛋白，融合到金属结合多肽的文库中，回收将通过离心法或磁力实现，并如前所述进行评估。

There are numerous biomedical and biotechnological applications for recombinant proteins, some of which, requires them to be stable under non-biological conditions. This is leading the demand for the immobilisation of proteins on surfaces. However, problems can arise due to the requirement for the surface material properties being preserved, as well as retaining the protein activity throughout the whole process of production, purification and surface binding. Additionally, when the phases of production are separated, this allows for unforeseen errors to occur. This project proposes to use a Pichia pastoris modular cloning (MoClo) toolkit as well as a Yeast MoClo toolkit, in combination with a statistical Design of Experiment (DoE) approach to produce a rapid, automatable system for protein-based multi-functional materials.The project has four main aims:I. Development of Pichia pastoris as an expression host to produce surface-binding proteins.II. Validate Pichia pastoris for the production of surface-binding proteins of relevance to the manufacture of multi-functional coatings.III. Address the problem of "one-factor-at-a-time" optimisation in standard bio-design practices.IV. Extend the range of enzymes and surface materials. This will include the use of metal nanoparticles as a surface because they have been demonstrated to be directly coated with proteins leading to stable metal-NPs which retain protein activity. The aims and objectives would be achieved through the following procedures. Firstly, using the MoClo Yeast Toolkit, in combination with the MoClo Pichia Toolkit, cassette plasmids would be constructed containing different promoters, secretion tags and fluorescent reporters and transformed into E. coli before being transformed into P. pastoris by electroporation. The plasmid cassette would be integrated into the genome by homologous recombination, increasing the stability compared to plasmid-based expression due to the lack of a stable plasmid system in P pastoris. Secretion efficiency of the cell would be assessed by measuring the total fluorescence, then by measuring fluorescence of the supernatant post cell removal by centrifugation. Cellulose-binding proteins (CBP), from the iGEM registry of characterised cellulose-binding domains, would be fused to a fluorescent reporter. The ability of this to bind to cellulose would be analysed with increasing levels of purity, such as with the cells in the media, cells removed via centrifugation, ion exchange chromatography and gel filtration chromatography.Streptomyces sp. chaplins would be tested as there are species, such as Streptomyces reticuli, containing genes encoding for proteins involved in cellulose binding. C2-chitin binding protein of Polistes dominula would be tested as the protein shows similarity to cellulose binding proteins and has the ability to bind to cellulose.A DoE approach would be taken where all relevant parameters of the production, purification and surface binding properties will be changed simultaneously. The benefit of DoE is that optimisation is more effectively searched with the effect of interactions identified, and assessed, independently. After a proof-of-principle DoE model for automated production of cellulose-binding proteins, the range of enzymes and surface materials can be explored. Metal-NP's would be interesting to investigate because they have high surface-area to volume ratios along with plasmonic or magnetic properties, allowing for high surface binding efficiency and simple detection and separation. We would produce metal-binding proteins fused to a library of metal binding peptides, recovery would be achieved by centrifugation or magnetically and evaluated as mentioned previously.