Easy Production of “Difficult Peptides” Using Cell‐Free Protein Synthesis and a New Methionine Analogue as a Latent Peptide Cleavage Site

Abstract

Aliphatic γ-chloro-α-amino acids incorporated in place of their canonical analogues through cell-free protein synthesis act as heat-labile linkers, offering a useful strategy for the straightforward production of target peptides as fusion proteins, from which the targets are readily released. Until now, the natural abundance of aliphatic amino acids in peptides has limited the scope of the method, as it leads to undesired cleavage sites in synthesized products, but here the authors report the development of a new cleavable chloro amino acid that incorporates in place of the relatively rare amino acid methionine, thus greatly expanding the scope of producible targets. This new strategy is employed for simplified peptide synthesis with a methionine-free fusion partner, allowing single-site incorporation of the cleavable linker for clean release and easy purification of the target peptide. Its utility is demonstrated through the straightforward preparation of two peptides reported to be challenging targets and not accessible through standard solid-phase chemical methodologies, as well as analogues.

Point-of-Care Peptide Hormone Production Enabled by Cell-Free Protein Synthesis

“…After CFPS, we only observed full-length expression of 4 out of the 20 tested hormones (Figure S1a). Consistent with previous observations, 35,36 successfully expressed peptide hormones (insulin A and B chains fused with leucine zipper heterodimers, 47 growth hormone, and leptin) tended to be the largest (>10 kDa) of the peptide hormones in the panel. The larger bands in the insulin A and B chain heterodimer lanes may be due to unintended dimerization or trimerization from the heterodimer fusions.…”

“…Protease degradation makes it difficult to produce small peptides in crude-extract based CFPS and recombinantly in E. coli. − …”

Point-of-care peptide hormone production enabled by cell-free protein synthesis

“…After CFPS, we only observed full-length expression of 4 out of the 20 tested hormones ( Figure S1a ). Consistent with previous observations (33,34) , successfully expressed peptide hormones (insulin A and B chains fused with leucine zipper heterodimers (45) , growth hormone, and leptin) tended to be the largest (>10 kDa) of the peptide hormones in the panel. The larger bands in the insulin A and B chain heterodimer lanes may be due to unintended dimerization or trimerization.…”

“…While these efforts have covered numerous therapeutics, peptide biologics have been underrepresented due to challenges in CFPS production. Protease degradation makes it difficult to produce small peptides in crudeextract based CFPS and recombinantly in E. coli (33)(34)(35).…”

“…While these efforts have covered numerous therapeutics, peptide biologics have been underrepresented due to challenges in CFPS production. Protease degradation makes it difficult to produce small peptides in crude-extract based CFPS and recombinantly in E. coli ( 33–35 ).…”