Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system

Yue Wu, Zhenling Cui, Yen-Hua Huang, Simon J. Veer, Andrey V. Aralov, Zhong Guo, Shayli V. Moradi, Alexandra O. Hinton, Jennifer R. Deuis, Shaodong Guo, Kai-En Chen, Brett M. Collins, Irina Vetter, Volker Herzig, Alun Jones, Matthew A. Cooper, Glenn F. King, David J. Craik, Kirill Alexandrov () and Sergey Mureev ()
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Yue Wu: The University of Queensland
Zhenling Cui: ARC Centre of Excellence in Synthetic Biology
Yen-Hua Huang: The University of Queensland
Simon J. Veer: The University of Queensland
Andrey V. Aralov: Russian Academy of Sciences
Zhong Guo: ARC Centre of Excellence in Synthetic Biology
Shayli V. Moradi: ARC Centre of Excellence in Synthetic Biology
Alexandra O. Hinton: The University of Queensland
Jennifer R. Deuis: The University of Queensland
Shaodong Guo: The University of Queensland
Kai-En Chen: The University of Queensland
Brett M. Collins: The University of Queensland
Irina Vetter: The University of Queensland
Volker Herzig: The University of Queensland
Alun Jones: The University of Queensland
Matthew A. Cooper: The University of Queensland
Glenn F. King: The University of Queensland
David J. Craik: The University of Queensland
Kirill Alexandrov: ARC Centre of Excellence in Synthetic Biology
Sergey Mureev: Queensland University of Technology

Nature Communications, 2022, vol. 13, issue 1, 1-17

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-021-27854-9

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