title: Production of Nα-acetylated thymosin α1 in Escherichia coli.
abstract: BACKGROUND
  Thymosin α1 (Tα1), a 28-amino acid Nα-acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining Nα-acetylation. In this study, we describe a novel production process for Nα-acetylated Tα1 in Escherichia coli.
  
  
  RESULTS
  To obtain recombinant Nα-acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis), and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic Nα-acetyltransferase) in Escherichia coli. After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols β-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da). The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated.
  
  
  CONCLUSIONS
  The present data demonstrate that Nα-acetylated Tα1 can be efficiently produced in recombinant E. coli. This bioprocess could be used as an alternative to chemosynthesis for the production of Tα1. The described methodologies may also be helpful for the biosynthesis of similar peptides.
authors.affiliation: Institute of Biotechnology, Academy of Military Medical Sciences, 20 DongDa Street, FengTai District, Beijing 100071, China.
authors.forename: Yuantao
authors.initials: Y
authors.lastname: Ren
authors.forename: Xueqin
authors.initials: X
authors.lastname: Yao
authors.forename: Hongmei
authors.initials: H
authors.lastname: Dai
authors.forename: Shulong
authors.initials: S
authors.lastname: Li
authors.forename: Hongqing
authors.initials: H
authors.lastname: Fang
authors.forename: Huipeng
authors.initials: H
authors.lastname: Chen
authors.forename: Changlin
authors.initials: C
authors.lastname: Zhou
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; C103076:DnaX protein, Bacteria; C471213:His-His-His-His-His-His; D007527:Isoenzymes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013947:Thymosin; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; D004258:DNA Polymerase III; D000077596:Thymalfasin