Shulong Li

RimJ is responsible for Nα-acetylation of thymosin α1 in Escherichia coli

Nα-Acetylation is one of the most common protein modifications in eukaryotes but a rare event in prokaryotes. Some endogenously Nα-acetylated proteins in eukaryotes are frequently reported not to be acetylated or only very partially when expressed in recombinant Escherichia coli. Thymosin α1 (Tα1), an Nα-acetylated peptide of 28 amino acids, displays a powerful general immunostimulating activity. Here, we revealed that a fusion protein of thymosin α1 and L12 is partly Nα-acetylated in E. coli. Through deletion of some Nα-acetyltransferases by Red recombination, we found that, when rimJ is disrupted, the fusion protein is completely unacetylated. The relationship of rimJ and Nα-acetylation of Tα1 was further investigated by gene rescue and in vitro modification. Our results demonstrate that Nα-acetylation of recombinant Tα1-fused protein in E. coli is catalyzed by RimJ and that fully acetylated Tα1 can be obtained by co-expressing with RimJ. This is the first description that an ectopic protein acetylation in bacterial expression systems is catalyzed by RimJ, a known prokaryotic Nα-acetyltransferase.

Production of Nα-acetylated thymosin α1 in Escherichia coli

BackgroundThymosin 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.ResultsTo 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.ConclusionsThe 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.