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

Yue Wu,Matthew A Cooper,Andrey V Aralov,Alexandra O Hinton,Shayli Varasteh Moradi ,Zhong Guo,Jennifer R Deuis,Shaodong Guo,Yen‐Hua Huang ,David J Craik,Glenn F King,Alun Jones,Simon J De Veer,Brett M Collins,Kirill Alexandrov,Kai‐En Chen ,Zhenling Cui,Irina Vetter,Volker Herzig,Sergey Mureev

doi:10.1038/s41467-021-27854-9

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.

Highlights

Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping
In agreement with the previous report[42], we found near-linear inverse relationship betweenpeptide’s molar yield and size in the range from 49 to 315 amino acids (Supplementary Table 1) suggesting the inactivation of some key translation component(s) or resource(s) rather than inefficient ribosome recycling to be the major factor limiting the productivity of resin-assisted E. coli S30-based cell-free system (Ec CFS) (Supplementary Table 1, Supplementary Notes 2 and 3 and Supplementary Fig. 3A, B)
Thrombin efficiently cleaved the Arg-Gly bond in the RGS motif without affecting alternative cleavage site(s) within the constrained core of disulfide-bonded peptide (Supplementary Fig. 13C and Supplementary Note 10). We propose that these two approaches for the removal of the RGS-tag would enable the preparation of near-native forms for the majority of peptides

Summary

Introduction

Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. 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 While the former is superior in oral bioavailability and the ability to access intracellular targets, they lag behind the latter in potency and selectivity and, as a result, in safety. Robust statistics provided by Venomics platform[27,33], the success rate of peptide refolding following heterologous expression[31] or chemical synthesis[26] suggest that under the universal folding conditions only ~half of the peptides could be produced in a soluble form This is consistent with the existence of two principal folding modes—“framework” and “collapsed”, representing either the hierarchic condensation of native-like elements or the slow flux through the off-path folding states[34], respectively. A generally applicable method to accommodate both folding trajectories for cost-effective parallel production of complex disulfide-constrained peptides is expected to facilitate their development into drug-like products[35]

Methods

Results

Conclusion