Generation of mature N?-terminal acetylated thymosin ? 1 by cleavage of recombinant prothymosin ?.

ABSTRACT: N(?)-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. N(?)-terminal acetylated thymosin ?1 (T?1), 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 N(?)-terminal acetylation of recombinant prothymosin ? (ProT?) in E. coli. Here we present a method for production of N(?)-acetylated T?1 from recombinant ProT?. The recombinant ProT? was cleaved by human legumain expressed in Pichia pastoris to release T?1 in vitro. The N(?)-acetylated T?1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant N(?)-acetylated T?1 was 3108.79 in, which is identical to the mass of N(?)-acetylated T?1 produced by total chemical synthesis. This mass corresponded to the nonacetylated T?1 mass with a 42 Da increment. The retention time of recombinant N(?)-acetylated T?1 and chemosynthetic N(?)-acetylated T?1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of an E. coli expression system for the production of recombinant human N(?)-acetylated T?1 and also will provide the basis for the preparation of recombinant acetylated peptides in E. coli.

SUBMITTER: Liu B 

PROVIDER: S-EPMC3830889 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature