1. Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system.
- Author
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Wu, Yue, Cui, Zhenling, Huang, Yen-Hua, de Veer, Simon J., Aralov, Andrey V., Guo, Zhong, Moradi, Shayli V., Hinton, Alexandra O., Deuis, Jennifer R., Guo, Shaodong, Chen, Kai-En, Collins, Brett M., Vetter, Irina, Herzig, Volker, Jones, Alun, Cooper, Matthew A., King, Glenn F., Craik, David J., Alexandrov, Kirill, and Mureev, Sergey
- Subjects
PEPTIDES ,PROTEINS ,THERMODYNAMIC control ,CONOTOXINS ,PROTEIN folding ,RAPID prototyping - Abstract
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. Generic approach for rapid prototyping is essential for the progress of synthetic biology. Here the authors modify the cell-free translation system to control protein aggregation and folding and validate the approach by using single conditions for prototyping of various disulfide-constrained polypeptides. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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