Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, 11724, USA.
Nat Commun. 2022 Jan 11;13(1):260. doi: 10.1038/s41467-021-27854-9.
Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli-based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment.
肽和蛋白质治疗的进展增加了对快速且具有成本效益的多肽原型制作的需求。虽然体外翻译系统非常适合快速和多路复用的多肽原型制作，但它们会导致翻译产物的错误折叠、聚集和二硫键错配。在这里，我们提出如果在无聚集和热力学控制的折叠环境中进行，体外产生的富含二硫键的肽和蛋白质可以有效地折叠。为此，我们修改了基于大肠杆菌的体外翻译系统，以允许通过亲和基质对翻译产物进行共翻译捕获。该过程减少了蛋白质聚集，并在热力学控制下实现了有生产力的氧化折叠和错误折叠状态的循环利用。在这项研究中，我们表明所开发的方法可能适用于原型制作各种具有二硫键限制的肽、具有非天然键的大环肽和抗体片段，其数量足以进行相互作用分析和生物活性评估。
