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

重组蛋白有许多生物医学和生物技术应用，其中一些应用要求它们在非生物条件下保持稳定。这导致了对表面固定蛋白质的需求。然而，在生产、纯化和表面结合的整个过程中，由于要求保持表面材料的性质，以及保持蛋白质的活性，可能会出现问题。此外，当生产阶段分开时，可能会出现不可预见的错误。本项目拟使用毕赤酵母模块化克隆（MoClo）工具包和酵母MoClo工具包，结合实验统计设计（DoE）方法，建立一个快速、自动化的蛋白质多功能材料系统。该项目有四个主要目标：1。毕赤酵母作为表达宿主生产表面结合蛋白的研究进展。验证毕赤酵母生产与多功能涂料生产相关的表面结合蛋白。解决标准生物设计实践中“一次一因素”优化的问题。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，因为它们已被证明可以直接被蛋白质包裹，从而产生稳定的金属nps，保持蛋白质活性。各项目的和目标将通过下列程序实现。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质粒，转化到大肠杆菌中，再通过电穿孔转化到pastoris中。质粒盒将通过同源重组整合到基因组中，由于在帕斯德酵母中缺乏稳定的质粒系统，因此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量上清的荧光。纤维素结合蛋白（CBP），从iGEM登记的特征纤维素结合域，将融合到荧光报告。这种与纤维素结合的能力将随着纯度的增加而分析，例如培养基中的细胞，通过离心、离子交换色谱和凝胶过滤色谱去除的细胞。链霉菌（Streptomyces sp. chaplins）将被测试，因为有一些物种，如网状链霉菌（Streptomyces reticuli），含有编码参与纤维素结合的蛋白质的基因。由于该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Polistes dominula的c2 -甲壳素结合蛋白进行测试。采用DoE方法，同时改变生产、纯化和表面结合性能的所有相关参数。DoE的好处是，通过独立识别和评估相互作用的效果，可以更有效地搜索优化。在自动化生产纤维素结合蛋白的原理验证DoE模型之后，可以探索酶和表面材料的范围。金属np的研究将是有趣的，因为它们具有高表面积与体积比以及等离子体或磁性能，允许高表面结合效率和简单的检测和分离。我们将生产金属结合蛋白与金属结合肽库融合，通过离心或磁力回收，并按照前面提到的方法进行评估。

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.