Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom.
GSK Vaccines Institute for Global Health Srl, Siena, Italy.
Microbiol Spectr. 2023 Jun 15;11(3):e0359422. doi: 10.1128/spectrum.03594-22. Epub 2023 Apr 10.
The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.
自转运蛋白分泌系统以前曾被用于在实验室规模上将异源蛋白靶向分泌到细菌细胞表面和细胞外环境中。该平台特别适用于生产“困难”的重组蛋白，这些蛋白在细胞内产生时可能会产生毒性作用。IrmA 就是这样一种蛋白。IrmA 是一种候选疫苗，以包涵体的形式产生，需要重折叠。在这里，我们描述了自转运蛋白系统在分泌一种工业相关蛋白（IrmA）中的用途和放大。构建了表达 IrmA 的质粒，使得自转运平台能够将 IrmA 分泌到培养上清液部分。自转运平台适合于 IrmA 的生产和纯化，其物理性质与在细胞质中产生的蛋白相当。在放大蛋白生产条件下进行 IrmA 的生产，从培养上清液中获得了 29.3mg/L 的 IrmA 产量，与当前工业过程的产量一致。重组蛋白生产是生物技术领域的重要组成部分。在这里，我们表明自转运平台是一种可行的方法，可用于在工业相关规模上生产、分泌和纯化“困难”生产的蛋白。使用自转运平台可以减少所需的下游处理操作的数量，从而缩短重组蛋白生产的开发时间并降低成本。
