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

Thymosin beta 4 (T beta 4) 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 T beta 4 at lower costs than with the use of chemical synthesis. In this research, we developed a method to produce rhT beta 4 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 rhT beta 4-Intein was constructed, in which rhT beta 4 was fused to the N-terminus of the smallest mini-intein Spl DnaX. The rhTb4 could be fully acetylated when the rhT beta 4-Intein was expressed in the engineering strain. After purification, the rhT beta 4-Intein fusion protein was induced with dithiothreitol (DTT) to release rhT beta 4 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 rhT beta 4 can reach 200 mg per litre. The N-acetylated rhT beta 4 could be stable at 2-8 degrees C for 24 months in PBS buffer without protein degradation and concentration change. The N-acetylated rhT beta 4 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.