Hongmei Dai

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.

Constructing Modified Protein-producing Escherichia coli Capable of Autohydrolysing Host Nucleic Acid During Cell Lysis

We used Red recombination system to construct a modified E. coli protein-producing strain capable of autohydrolysing host nucleic acid. E. coli BL21 (DE3), a common protein-producing strain, was used as starting material. The modified E. coli expression host had a staphylococcal nuclease expression cassette within the host chromosome lpxM locus. The Staphylococcus aureus nuclease was expressed as a fusion to the ompA signal peptide, and was translocated to the periplasm of the cell, protecting the host nucleic acid from the toxic activity during growth. The nuclease was released during cell lysis and subsequently hydrolyzed host nucleic acid in the lysate. Results show that the modified strain had sufficient nuclease activity to completely autohydrolyze the host chromosomal DNA and produced same amount of recombinant proteins as the original strain.

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

Abstract Thymosin β4 (Tβ4) is a multifunctional N-acetylated peptide with distinct activities important at various stages. Due to its potential multiple therapeutic uses in many fields, there is an increasing need of Tβ4 at lower costs than with the use of chemical synthesis. In this research, we developed a method to produce rhTβ4 with N-acetylation in E. coli. Firstly, the E. coli strain whose chromosome being integrated by the specific N-terminal acetyltransferase ssArd1 was constructed. Secondly, the rhTβ4-Intein was constructed, in which rhTβ4 was fused to the N-terminus of the smallest mini-intein Spl DnaX. The rhTβ4 could be fully acetylated when the rhTβ4-Intein was expressed in the engineering strain. After purification, the rhTβ4-Intein fusion protein was induced with dithiothreitol (DTT) to release rhTβ4 through intein-mediated N-terminal cleavage. Under the optimal conditions, the N-terminal serine residue was shown to be 100% acetylated and the yield of N-acetylated rhTβ4 can reach 200 mg per litre. The N-acetylated rhTβ4 could be stable at 2–8 °C for 24 months in PBS buffer without protein degradation and concentration change. The N-acetylated rhTβ4 also showed the activity of binding with actins from different sources and excellent therapeutic effect on the rats with moderate to severe dry eye disease.