Intein-mediated expression, purification, and characterization of thymosin α1–thymopentin fusion peptide in Escherichia coli

Thymosin α1–thymopentin (Tα1–TP5) fusion peptide has been proved to be an immune regulator based on its higher immunoregulatory activity than Tα1 and TP5. To obtain Tα1–TP5 more effectively and economically, Tα1–TP5 was genetically fused to a self-cleaving intein-chitin binding domain tag for purification via chitin beads in Escherichia coli. After affinity purification, the target peptide was released from the chitin beads via self-cleaving intein ((INTervening protEIN) induced by dithiothreitol. Further, Tα1–TP5 was purified by Superdex 30 and identified by Tricine-SDS-PAGE and electrospray ionization-mass spectrometry. Finally, about 7.6 mg Tα1–TP5 purified from the soluble fraction and inclusion bodies was obtained from 1 L culture media. The purity was 95% after a series of chromatographic purification steps. In vitro, the purified Tα1–TP5 could stimulate the proliferation of mouse splenic lymphocytes. Overall, this work demonstrated that Tα1–TP5 was purified with low cost and high efficiency, greatly expanding its potential use as an immune regulator.

► Thymosin α1–thymopentin (Tα1–TP5) fusion peptide was firstly expressed in Escherichia coli. ► Tα1–TP5 was purified from the soluble fraction and inclusion bodies of Escherichia coli by intein mediated self-cleavage. ► Purified Tα1–TP5 has higher activity than Tα1 in promoting lymphocyte proliferation.