title: Generation of mature Nα-terminal acetylated thymosin α 1 by cleavage of recombinant prothymosin α.
abstract: N(α)-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. N(α)-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 N(α)-terminal acetylation of recombinant prothymosin α (ProTα) in E. coli. Here we present a method for production of N(α)-acetylated Tα1 from recombinant ProTα. The recombinant ProTα was cleaved by human legumain expressed in Pichia pastoris to release Tα1 in vitro. The N(α)-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant N(α)-acetylated Tα1 was 3108.79 in, which is identical to the mass of N(α)-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 N(α)-acetylated Tα1 and chemosynthetic N(α)-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 N(α)-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides in E. coli.
authors.affiliation: Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.forename: Xin
authors.initials: X
authors.lastname: Gong
authors.forename: Shaohong
authors.initials: S
authors.lastname: Chang
authors.forename: Peng
authors.initials: P
authors.lastname: Sun
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism
chemical_list: D011498:Protein Precursors; D011994:Recombinant Proteins; C040782:prothymosin alpha; D013947:Thymosin; D000077596:Thymalfasin