Same Day Access to Proteins Enabled by Folding Selections

Abstract

From laundry detergent to biomedicines, proteins are core components of modern technology. However, despite significant progress, modern recombinant protein expression still requires days to months to deliver pure, functional material. Chemical protein synthesis can also afford proteins for study with similar timeframes at the chemistry stage, with additional time dedicated post-synthesis for handling as separation of the numerous closely related side-products requires weeks to months. To overcome these rate-limiting barriers, we propose folding selection as a new framework to understand synthetic side products and enable rapid purification. We demonstrate that the minor chemical modifications present on synthetic side products result in substantially altered physical-chemical properties and that simple bio-purification techniques can separate them from the native protein in hours. With this strategy, we demonstrate the production of nine functional synthetic proteins in under ten hours each, including disulfide-containing enzymes and transcription factor domains with non-natural amino acids. Furthermore, only possible with this technology, we access homogeneous, post-translationally modified (e.g., by phosphorylation or acetylation) proteins in milligram amounts in hours. Understanding protein function is a cornerstone of modern biology, and the same-day protein production described here is uniquely suited to accelerate these efforts.

Rapid flow-based synthesis of post-translationally modified peptides and proteins: a case study on MYC's transactivation domain

“…2-3 min per residue. [27][28][29] While ow-based protein synthesis has been successfully used to install single PTMs (phosphorylation, acetylation) into a protein, 30 the routine incorporation of several PTM amino acid building blocks into a single sequence has not yet been reported. Phosphorylation and polyphosphorylation, in particular, have posed a signicant challenge in chemical peptide and protein synthesis due to difficult couplings (bulky side-chain protecting groups) and the occurrence of side-reactions (belimination of the phosphate) during SPPS, leading to large amounts of undesired side-products and low yields.…”

Rapid Flow-Based Synthesis of Post-Translationally Modified Peptides and Proteins: A Case Study on MYC’s Transactivation Domain

“…2-3 min per residue. [27][28][29] While flow-based protein synthesis has been successfully used to install single PTMs (phosphorylation, acetylation) into a protein, 30 the routine incorporation of several PTM amino acid building blocks has not yet been reported. Phosphorylation and polyphosphorylation, in particular, have posed a significant challenge in chemical peptide and protein synthesis due to difficult couplings (bulky side-chain protecting groups) and the occurrence of side-reactions (ß-elimination of the phosphate) during SPPS, leading to large amounts of undesired side-products and low yields.…”