Selective biochemical and synthetic biology approaches for improved delivery of recombinant proteins to the extracellular milieu

用于改善重组蛋白向细胞外环境的递送的选择性生化和合成生物学方法

• 批准号: BB/I020756/1
• 负责人: Ian Henderson
• 金额: $ 56.59万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI020756%2F1
• 关键词: Selective; biochemical; synthetic; biology; approaches

The biotechnology industry produces revenues in excess of £500 billion per year. This industry relies on the production of proteins to produce biopharmaceuticals (novel drugs and vaccines), bioproducts for use in food production and in foods (animal health-care biologics, biological plant-growth promoters and pesticides, nutritional supplements, and food additives), bioremediation to clean up the environment (e.g. enzymes to break down oil after oil spillages) and may offer alternatives to environmentally polluting or fossil-fuel-consuming manufacturing processes (e.g. methods to produce hydrogen from bacteria; industrial catalysts). The standard mechanism for making proteins is to produce them in a non-pathogenic bacterium called E. coli. However, there are limits to the types of protein that can be produced in E. coli. As most of these proteins are not derived from E. coli and are produced at levels much greater than E. coli would normally produce any natural protein, the E. coli bacterium often deals with them by storing the proteins in an inactive form in a compartment of the cell called the inclusion body. The accumulation of proteins in inclusion bodies causes problems for commercial production, including expensive steps to recover the proteins in an active conformation. To overcome these problems some proteins are produced in another compartment of the cell called the periplasm. However, if too much protein is made in the periplasm it can burst the cell open, killing the cell in the process, and therefore little or no protein is produced. Even those that are stable when produced in the periplasm will need expensive purification as there are many other native E. coli proteins in this part of the cell. When expressing a non-native protein the best place for the E. coli bacterium to place the protein is outside of the cell - this prevents accumulation of the protein in inclusion bodies or in the periplasm which might lead to cell death. In addition, the laboratory strains of E. coli produce none, or very little, native proteins outside of the cell. Thus, when proteins are produce outside the cell they are relatively clean and need much less effort and investment to bring them to purity. However, for a number of technical reasons it has proved to be very difficult to produce proteins outside the bacterial cell. Here we will utilise a very simple system called the Autotransporter system which has a proven track record for producing non-native proteins outside of the cell. We have already used this system to produce a proteins relevant to industry proving the system works. We will now attempt to make the system produce proteins at levels which are beneficial to industry.

生物技术产业每年产生超过5000亿英镑的收入。该行业依靠生产蛋白质来生产生物药品（新药和疫苗）、食品生产和食品中使用的生物产品（动物保健生物制剂、生物植物生长促进剂和农药、营养补充剂和食品添加剂）。清洁环境的生物修复（例如，在石油泄漏后分解石油的酶），并可能为污染环境或消耗化石燃料的制造过程（例如，从细菌中产生氢的方法；工业催化剂）提供替代方案。制造蛋白质的标准机制是在一种叫做大肠杆菌的非致病性细菌中产生蛋白质。然而，大肠杆菌中可以产生的蛋白质类型是有限的。由于这些蛋白质中的大多数不是来自大肠杆菌，其产生的水平远远高于大肠杆菌通常产生的任何天然蛋白质，因此大肠杆菌通常通过将蛋白质以非活性形式储存在称为包涵体的细胞隔间中来处理它们。包涵体中蛋白质的积累给商业生产带来了问题，包括恢复活性构象的昂贵步骤。为了克服这些问题，一些蛋白质在细胞的另一个称为周质的隔室中产生。然而，如果在周质中产生过多的蛋白质，它会使细胞破裂，在这个过程中杀死细胞，因此很少或根本不产生蛋白质。即使是那些在周质中产生的稳定蛋白也需要昂贵的纯化，因为在细胞的这一部分还有许多其他天然的大肠杆菌蛋白。当表达非天然蛋白质时，大肠杆菌放置蛋白质的最佳位置是在细胞外，这可以防止蛋白质在包涵体或周质中积聚，从而可能导致细胞死亡。此外，大肠杆菌的实验室菌株在细胞外不产生或只产生很少的天然蛋白质。因此，当蛋白质在细胞外产生时，它们是相对清洁的，需要更少的努力和投资来达到纯度。然而，由于一些技术原因，在细菌细胞外生产蛋白质被证明是非常困难的。在这里，我们将利用一种非常简单的系统，称为Autotransporter系统，它在细胞外产生非天然蛋白质方面有良好的记录。我们已经用这个系统生产了一种与工业相关的蛋白质，证明了这个系统是有效的。我们现在将尝试使该系统生产对工业有益的蛋白质。

项目成果

Mutational and topological analysis of the Escherichia coli BamA protein.

• DOI: 10.1371/journal.pone.0084512
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Browning DF;Matthews SA;Rossiter AE;Sevastsyanovich YR;Jeeves M;Mason JL;Wells TJ;Wardius CA;Knowles TJ;Cunningham AF;Bavro VN;Overduin M;Henderson IR
• 通讯作者: Henderson IR