Synthesis of proteins by automated flow chemistry

Abstract

Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

Deep Learning for Prediction and Optimization of Fast-Flow Peptide Synthesis

“…Integral, width and height of the time-resolved traces were obtained using a modified version of the earlier published code. (12) Deep learning and optimization Data pre-processing. The data set obtained from the AFPS was pre-processed before analysis (SI Section 3.1).…”

“…(6) In addition, we recently demonstrated the advantages of automated fast-flow peptide synthesis (AFPS) over traditional SPPS techniques in terms of higher synthetic fidelity, increased length of the peptide chains accessible, and significant decrease in synthesis time. (12) Advancements in computational methods allow for the investigation of large-scale problems and previously inaccessible correlations in organic reaction methodology. Improved algorithms can predict reactivity and plan retrosynthetic routes from data.…”

Flow‐based SPPS for protein synthesis: A perspective

“…The content of d ‐amino acid was dependent on the protecting group, temperature and flow rate (and therefore the activation time) and independent of coupling agent (HATU/[7‐azabenzotriazol‐1‐yloxy]trispyrrolidinophosphonium hexafluorophosphate, PyAOP) and total coupling cycles. [ 12,14 ] Activation of Fmoc‐Cys(Trt)‐OH and Fmoc‐His(Boc)‐OH with PyAOP at 60°C through a shorter 5′ preheat‐activation loop resulted in minimal d ‐amino acid content. In addition, synthesis on the AFPS showed an improvement for the synthesis of “difficult peptides”, which can likely be attributed to the high synthesis temperature.…”

“…In this review, we highlight recent work that tests the limits of flow‐based Fmoc‐chemistry SPPS for the stepwise synthesis of single‐domain proteins, [ 14 ] and the historical background necessary to place this work in context. We examine the origins of flow‐based SPPS, especially with respect to their motivation and the challenges faced by early adopters, and identify the use of elevated temperature as a recent advance with considerable potential for impact.…”

Photocatalytic methods for amino acid modification

“…This method can easily facilitate the introduction of non-canonical amino acids (ncAAs), and while this was previously limited to peptides of less than 50 AA, recent developments have drastically increased that limit. 2 Alternatively, larger peptides and proteins may be produced by in vitro cellular expression, by introduction of the encoding DNA sequence into the expression host. While large proteins are more easily produced using this method, introduction of ncAAs in vitro requires the use of codon-suppression technology and addition of the ncAA to the cell suspension.…”