Generation of Mature Nα-Terminal Acetylated Thymosinα1 by Cleavage of Recombinant Prothymosinα

Abstract

Nα-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. Nα-terminal acetylated thymosinα1 (Tα1), a 28-amino-acid peptide, is an immune modifier that has been used in the clinic to treat hepatitis B and C virus (HBV/HCV) infections. We previously documented Nα-terminal acetylation of recombinant prothymosinα(ProTα) inE. coli. Here we present a method for production of Nα-acetylated Tα1 from recombinant ProTα. The recombinant ProTαwas cleaved by human legumain expressed inPichia pastoristo release Tα1in vitro. The Nα-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant Nα-acetylated Tα1 was 3108.79 in, which is identical to the mass of Nα-acetylated Tα1 produced by total chemical synthesis. This mass corresponded to the nonacetylated Tα1 mass with a 42 Da increment. The retention time of recombinant Nα-acetylated Tα1 and chemosynthetic Nα-acetylated Tα1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of anE. coliexpression system for the production of recombinant human Nα-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides inE. coli.