Design of a substrate-tailored peptiligase variant for the efficient synthesis of thymosin-α₁

Abstract

The synthesis of thymosin-α, an acetylated 28 amino acid long therapeutic peptide, via conventional chemical methods is exceptionally challenging. The enzymatic coupling of unprotected peptide segments in water offers great potential for a more efficient synthesis of peptides that are difficult to synthesize. Based on the design of a highly engineered peptide ligase, we developed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the production of thymosin-αvia a 14-mer + 14-mer segment condensation strategy. Using structure-inspired enzyme engineering, the thiol-subtilisin variant peptiligase was tailored to recognize the respective 14-mer thymosin-α segments in order to create a clearly improved biocatalyst, termed thymoligase. Thymoligase catalyzes peptide bond formation between both segments with a very high efficiency (>94% yield) and is expected to be well applicable to many other ligations in which residues with similar characteristics (e.g. Arg and Glu) are present in the respective positions P1 and P1'. The crystal structure of thymoligase was determined and shown to be in good agreement with the model used for the engineering studies. The combination of the solid phase peptide synthesis (SPPS) of the 14-mer segments and their thymoligase-catalyzed ligation on a gram scale resulted in a significantly increased, two-fold higher overall yield (55%) of thymosin-α compared to those typical of existing industrial processes.

Sustainability in peptide chemistry: current synthesis and purification technologies and future challenges

“…192 In this context, the subtilisin variants developed by Enzypep have been used for the synthesis of at least two therapeutic peptides, namely exenatide and thymosin-α. 193,194…”

Subtiligase-Catalyzed Peptide Ligation

“…Subtiligase therefore represents a practical tool for peptide segment condensation under kinetic control. Since its development, subtiligase has found widespread application in many areas of chemistry and biology, including peptide synthesis and cyclization, , total protein synthesis, site-specific protein bioconjugation, , protein semisynthesis, and mapping proteolytic cleavage sites, among others. The initial S221C/P225A design has also served as a starting point for engineering many of subtiligase’s enzymatic properties, including substrate specificity and ligation-to-hydrolysis ratio. , …”

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology

“…Apart from the posttranslational modification via N-terminal acetylation, chemical synthesis of Tα1 is complex, and obstacles comprise the large number of required protecting groups as well as the aggregation tendency of intermediates during synthesis [24]. Furthermore, the overall yield of the solid-phase synthesis is low, typically reaching only around 25% [25]. On the other hand, the biotechnological production as a recombinant peptide in an economic manner has failed so far.…”