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

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, Mureev, Sergey (January 2022) Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system. Nature Communications, 13 (1). p. 260. ISSN 2041-1723

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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.

Item Type:	Paper
Subjects:	bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification organism description > animal > insect > Drosophila bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification organism description > animal organism description > bacteria organism description > bacteria > escherichia coli organism description > animal > insect bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression
CSHL Authors:	Wu, Yue
Communities:	CSHL labs > Borniger lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	11 January 2022
Date Deposited:	21 Jul 2022 13:24
Last Modified:	17 Jan 2024 19:20
PMCID:	PMC8752827
URI:	https://repository.cshl.edu/id/eprint/40680

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