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Biotechnological production of acetylated thymosin β4

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Abstract

Thymosin β4 (43 aa) is a highly conserved acidic peptide, which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin β4 is undergoing clinical trials as a drug for treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin β4 is produced by a solid-phase chemical synthesis. Biotechnological synthesis of this peptide is difficult, because the N-terminal amino acid residue of thymosin β4 playing an essential role in the actin interaction is acetylated. In this study, we proposed a method for production of a thymosin β4 recombinant precursor and its directed chemical acetylation. Deacetylthymosin β4 was synthesized as a part of a hybrid protein containing thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for purification of deacetylthymosin β4: (i) biosynthesis of a soluble hybrid protein (HP) in Escherichia coli, (ii) isolation of HP by ion exchange chromatography, (iii) cleavage of HP with TEV protease, and (iv) purification of deacetylthymosin β4 by ultrafiltration. N-Terminal acetylation of the serine residue of deacetylthymosin β4 was performed with acetic anhydride under acidic conditions (pH 3.0). The reaction yield was 55%. Thymosin β4 was finally purified by reverse-phase HPLC. The proposed method of isolation of recombinant thymosin β4 can be scaled-up and provide a highly purified preparation in a yield of 20 mg per 1 L of culture suitable for use in medical practice.

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Abbreviations

DDT:

D,L-dithiothreitol

HP:

hybrid protein

IPTG:

isopropyl-β-D-thiogalactopyranoside

PMSF:

phenylmethylsulfonyl fluoride

Tβ4:

thymosin β4

TEV:

tobacco etch virus

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia

    K. A. Beyrakhova, V. N. Stepanenko, A. I. Miroshnikov & R. S. Esipov

Authors
  1. K. A. Beyrakhova
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  2. V. N. Stepanenko
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  3. A. I. Miroshnikov
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  4. R. S. Esipov
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Corresponding author

Correspondence to R. S. Esipov.

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Original Russian Text © K.A. Beyrakhova, V.N. Stepanenko, A.I. Miroshnikov, R.S. Esipov, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 2, pp. 223–232.

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Beyrakhova, K.A., Stepanenko, V.N., Miroshnikov, A.I. et al. Biotechnological production of acetylated thymosin β4. Russ J Bioorg Chem 37, 198–206 (2011). https://doi.org/10.1134/S1068162011020026

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  • DOI: https://doi.org/10.1134/S1068162011020026

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