Production of N α -acetylated thymosin α1 in Escherichia coli

Abstract

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.

Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700

“…However, because chemical synthesis is cost-intensive, an efficient biosynthetic approach would be better suited for CAM-W production. Several recombinant small peptides have been successfully expressed in E. coli 89 and Pichia pastoris 1011 expression systems. However, the expression of heterologous proteins in E. coli often results in inclusion bodies that do not exhibit any biological activity and requires solubilization, refolding and purification procedures to recover functionally active products12.…”

Highly effective biosynthesis of N-acetylated human thymosin β4 (Tβ4) in Escherichia coli

“…Both sides of the targeting fragment containing kanamycin resistant gene and ssArd1 had restriction sites of I-SceI, and homologous arms were assembled upstream and downstream about 1000 bp of the target gene. According to our reported method [33], the targeting plasmid and the helper plasmid were co-transformed into E. coli BL21 (DE3) and the Red recombinase and I-SceI then coexpressed by being induced by 0.2% L-Arabia sugar. I-SceI could cut off the target DNA fragments from the plasmid pBRSS-LPLR-Kan-Ard1.…”

“…The construction of plasmid pET-Intein which expressions the gene of the intein-Spl DnaX has been described before [33]. Briefly, the gene that encodes the Spl DnaX intein (136 amino acid residues; from Spirulina platensis) contained EcoRI and XhoI restriction enzyme sites was designed with the E. coli codon bias and cloned into the pET22b (þ) vector.…”

“…The tissue sections were fixed in 4% paraform-aldehyde and embedded in paraffin. The HE staining was performed as described elsewhere [33] to observe the changes in corneal histopathology. The sections were also stained with Periodic acid-Schiff reagent.…”

RimJ-Catalyzed Sequence-Specific Protein N-Terminal Acetylation in <i>Escherichia coli</i>

“…Interestingly, either the presence or absence of the N α -acetyl group is critical for the activities of some of these biologically important proteins. For example, Tα is active when N α -acetylated but TIMPs are inactive when N α -acetylated [12] [23].…”