Development of cell-free protein synthesis platforms for disulfide bonded proteins
Development of cell-free protein synthesis platforms for disulfide bonded proteins
复制标题
DOI：
10.1002/bit.21567
复制
发表时间：
2008-02-01
影响因子：
3.8
通讯作者：
Swartz, James R.
中科院分区：
文献类型：
--
作者：
Goerke, Aaron R.;Swartz, James R.
利用无细胞蛋白合成（CFPS）生产重组蛋白是一项重要的技术。例如，无细胞系统的开放性允许控制反应环境，以促进二硫键结合的蛋白质以快速且经济可行的形式折叠。这些优点使得无细胞蛋白质表达系统特别适合于产生患者特异性治疗性疫苗或解毒剂，以响应来自天然和人造生物制剂的威胁，以及用于难以在活细胞中产生的药物蛋白质。在这项工作中，我们评估了现代无细胞方法的多功能性，优化表达和折叠参数，并强调了合理设计的质粒模板在我们的大肠杆菌中生产哺乳动物分泌蛋白，融合蛋白和抗体片段的重要性。基于大肠杆菌的UPS系统。通过开发标准化提取物制备方案和通用无细胞反应条件，建立了两个独特的UPS平台。通用反应条件使所有蛋白质都能很好地表达，最佳治疗性蛋白质产率为710 μ g/mL，抗体片段产率为230 μ g/mL，疫苗融合蛋白产率为300 μ g/mL;大多数蛋白质正确折叠。当无细胞反应条件针对每种蛋白质进行优化时，获得了更好的产率。建立通用UPS平台增强了无细胞蛋白质合成的潜力，以低生产和资本成本可靠地生产复杂的蛋白质产品，并具有非常快速的工艺开发时间表。
The use of cell-free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make cell-free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell-free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli-based UPS system. Two unique UPS platforms were established by developing standardized extract preparation protocols and generic cell-free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 mu g/mL, an antibody fragment at 230 mu g/mL, and a vaccine fusion protein at 300 mu g/mL; with the majority correctly folded. Better yields were obtained when cell-free reaction conditions were optimized for each protein. Establishing general UPS platforms enhances the potential for cell-free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines.