title: Development of the intein-mediated method for production of recombinant thymosin β4 from the acetylated in vivo fusion protein.
abstract: Thymosin β4 is a 43 amino acid long peptide with an acetylated N-terminal serin that has a high potential as a remedy for healing ulcers, wounds and burns. Although protein biosynthesis offers attractive opportunities in terms of a large-scale production, currently thymosin β4 is mainly produced by chemical synthesis. The problems that hinder the successful commercialization of the biotechnological approach are associated with the small peptides expression and N-terminal acetylation. This work presents an innovative biotechnological method for thymosin β4 production that employs the peptide acetylation in vivo. A genetically engineered construct was created, where the Tβ4 coding sequence fused with the intein Mxe GyrA sequence and chitin-binding domain was combined with the acetyltransferase coding sequence to form a polycistronic construct under a stringent control of T7 promoter. This plasmid construct provided for the expression of the Tβ4-intein fusion protein. In the process of the post-translational modification in vivo formyl methionine was completely removed from the target peptide N-terminus and followed by the Tβ4 precursor N-terminal acetylation. The use of the intein-mediated expression system made it possible to extract thymosin β4 in only 2 chromatographic runs. The method is straightforward to implement and scale up.
authors.affiliation: M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, GSP-7, Miklukho-Maklaya Str. 16/10, 117997 Moscow, Russian Federation. Electronic address: esipov@ibch.ru.
authors.forename: Roman S
authors.initials: RS
authors.lastname: Esipov
authors.affiliation: M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, GSP-7, Miklukho-Maklaya Str. 16/10, 117997 Moscow, Russian Federation. Electronic address: youngchemist@mail.ru.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Makarov
authors.affiliation: M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, GSP-7, Miklukho-Maklaya Str. 16/10, 117997 Moscow, Russian Federation. Electronic address: svn@ibch.ru.
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Stepanenko
authors.affiliation: M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, GSP-7, Miklukho-Maklaya Str. 16/10, 117997 Moscow, Russian Federation. Electronic address: aiv@ibch.ru.
authors.forename: Anatoly I
authors.initials: AI
authors.lastname: Miroshnikov
mesh_terms: D000107:Acetylation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D047668:Inteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013947:Thymosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000099201:Thymosin beta(4)
chemical_list: D011993:Recombinant Fusion Proteins; D013947:Thymosin; D000099201:Thymosin beta(4)
keywords: Fusion protein; Intein; Protein acetylation; RimJ; RimL; Thymosin beta 4