title: Intein-mediated expression, purification, and characterization of thymosin α1-thymopentin fusion peptide in Escherichia coli.
abstract: 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.
authors.affiliation: Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
authors.forename: Pingli
authors.initials: P
authors.lastname: Li
authors.forename: Fengshan
authors.initials: F
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D047668:Inteins / Q000235:genetics*; D008214:Lymphocytes; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D000077596:Thymalfasin; D016305:Thymopentin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013947:Thymosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D013947:Thymosin; D016305:Thymopentin; D000077596:Thymalfasin