Generation of Mature Nα-Terminal Acetylated Thymosin α1 by Cleavage of Recombinant Prothymosin α

Abstract EN

Nα-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes.

Nα-terminal acetylated thymosin α1 (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 prothymosin α (ProTα) in E.

coli.

Here we present a method for production of Nα-acetylated Tα1 from recombinant ProTα.

The recombinant ProTα was cleaved by human legumain expressed in Pichia pastoris to release Tα1 in 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 an E.

coli expression system for the production of recombinant human Nα-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides in E.

coli.