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

The manufacture of long peptides using conventional chemical strategies is still very challenging and the technology used has remained almost unchanged since Merrifield developed the solid phase approach in the 1960s. Solid-phase peptide synthesis (SPPS) is characterized by an exponential decrease in the crude yield and purity as the peptide length increases: coupling and deprotection steps become less efficient and purification from accumulating by-products becomes increasingly difficult. In contrast, segment condensation processes are intrinsically more efficient, since short peptide segments can be produced in high yield and purity. However, the chemical ligation of peptide segments is still a significant challenge, mainly due to the low solubility of the protected segments and the potential epimerization of the C-terminal amino acid upon activation. To overcome some of these difficulties, the application of chemo-enzymatic peptide synthesis (CEPS), a combination of conventional SPPS for the production of unprotected peptide segments and an enzymatic epimerization-free coupling of the segments in water, represents a promising strategy. Hence, enzyme-mediated ligation technologies, e.g. the use of sortases, butelase-1, trypsiligase and subtilisin variants such as subtiligase or peptiligases, have recently gained increased attention for a more cost-efficient synthesis of medium-sized or long peptide.
A well-known peptide that is difficult to synthesize using conventional methodologies is thymosin-α1, an acetylated 28-mer therapeutic peptide (Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN-OH, 3108.32 g mol−1) with immunoregulating activity. 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-α1via 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-α1 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-α1 compared to those typical of existing industrial processes.