Abstract
Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.
Highlights
Thymosin α1 (Tα1) is an immunostimulatory peptide initially isolated from calf thymus [1] and abundant in humans
Our study provides the basis for therapeutic development of a generation thymosin α1 with prolonged circulation
To achieve C-terminal PASylation of N-terminally acetylated Tα1, a plasmid harboring a bicistronic operon was constructed to allow the simultaneous expression of human
Summary
Thymosin α1 (Tα1) is an immunostimulatory peptide initially isolated from calf thymus [1] and abundant in humans. Cleaved by the lysosomal asparaginyl endopeptidase (legumain; δ-secretase), the N-terminally acetylated 28-residue peptide Tα1 gets released [4]. T helper (Th) cells and provokes a shift towards the Th1 subclass, promoting the cell-mediated immune response. Tα1 mediates increased intracellular glutathione (GSH) levels [11], which inhibits the growth of certain cancer cells in vitro [7,12], and blocks the assembly of virus particles by hindering disulfide bond formation that is required for envelope glycoprotein oligomerization [13]
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