title: [Biotechnological production of acetylated thymosin beta4].
abstract: Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.
authors: K A
authors: KA
authors: Beĭrakhova
authors: V N
authors: VN
authors: Stepanenko
authors: A I
authors: AI
authors: Miroshnikov
authors: R S
authors: RS
authors: Esipov
mesh_terms: D000089:Acetic Anhydrides / Q000737:chemistry; D000107:Acetylation; D001709:Biotechnology / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003316:Corneal Diseases / Q000188:drug therapy*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009203:Myocardial Infarction / Q000188:drug therapy*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D013947:Thymosin / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use*; D014647:Varicose Ulcer / Q000188:drug therapy*; D000099201:Thymosin beta(4)
chemical_list: D000089:Acetic Anhydrides; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D013947:Thymosin; D000099201:Thymosin beta(4); C031800:acetic anhydride