Advertisement

Russian Journal of Bioorganic Chemistry

, Volume 44, Issue 1, pp 32–40 | Cite as

Biotechnological Method for Production of Recombinant Peptide Analgesic (Purotoxin-1) from Geolycosa sp. Spider Poison

  • R. S. Esipov
  • V. N. Stepanenko
  • I. O. Zvereva
  • D. A. Makarov
  • M. A. Kostromina
  • T. I. Kostromina
  • T. I. Muravyova
  • A. I. Miroshnikov
  • E. V. Grishin
Article

Abstract

Severe chronic and sometimes incurable diseases are frequently accompanied by a pain syndrome. Purotoxin-1, isolated from the poison of the Central Asian Geolycosa sp. spider and selectively inhibiting the purinergic P2X3 receptor (which is considered as a target for the control of the pain states), is one potentially highly effective drug with an analgesic effect. To produce the recombinant purotoxin-1, we created four genetically engineered constructions with different carrier proteins for the expression in E. coli: pTRX-PT1, pCBD-PT1, pGyrA-PT1, pDnaB-PT1. The construction with mini-intein DnaB from the Synechocystis sp. was the most efficient. Using the E. coli C3030/pDnaB-PT1 producer strain, the laboratory method, based on which a pilot technology of recombinant purotoxin-1 production was developed as a result of optimization and scaling, was created. Six grams of recombinant PT1 preparation with the confirmed pharmacological purity was developed for preclinical trials.

Keywords

production of recombinant proteins intein analgesic peptide toxin 

Abbreviation

ATP

adenosine triphosphate

CBD

chitinbinding domain

GyrA

gyrase A

EDTA

ethylenediaminetetraacetic acid

IPTG

isopropyl-β-D-1-thiogalactopyranoside

MES

2-(N-morpholino)ethanesulfonic acid

PMSF

phenylmethylsulfonyl fluoride

PT1

purotoxin-1

TRX

thioredoxin

ppm

part per million

rpHPLC

reversed-phase highperformance liquid chromatography

EU

endotoxin unit

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Grishin, E.V., Savchenko, G.A., Vassilevski, A.A., Korolkova, Y.V., Boychuk, Y.A., Viatchenko-Karpinski, V.Y., Nadezhdin, K.D., Arseniev, A.S., Pluzhnikov, K.A., Kulyk, V.B., Voitenko, N.V., and Krishtal, O.O., Ann. Neurol., 2010, vol. 67, no. 5, pp. 680–683.PubMedGoogle Scholar
  2. 2.
    Andreev, Y.A., Vassilevski, A.A., and Kozlov, A.S., Recent Pat. Inflamm. Allergy Drag. Discov., 2012, vol. 6, no. 1, pp. 35–45.CrossRefGoogle Scholar
  3. 3.
    Kabanova, N.V., Vassilevski, A.A., Rogachevskaja, O.A., Bystrova, M.F., Korolkova, Y.V., Pluzhnikov, K.A., Romanov, R.A., Grishin, E.V., and Kolesnikov, S.S., Biochim. Biophys. Acta, 2012, vol. 1818, pp. 2868–2875.CrossRefPubMedGoogle Scholar
  4. 4.
    Vasilevskii, A.A., Savchenko, G.A., Korol’kova, Yu.V., Boichuk, Ya.A., Pluzhnikov, K.A., Kryshtal’, O.A., and Grishin, E.V., Peptide modulator of purinergic receptors, RF Patent no. 2422459, Byull. Izobret., 2011, no. 18.Google Scholar
  5. 5.
    Gasparian, M.E., Bobik, T.V., Kim, Y.V., Ponomarenko, N.A., Dolgikh, D.A., Gabibov, A.G., and Kirpichnikov, M.P., Biochimie, 2013, vol. 95, no. 11, pp. 2076–2081.CrossRefPubMedGoogle Scholar
  6. 6.
    Beirakhova, K.A., Stepanenko, V.N., Miroshnikov, A.I., and Esipov, R.S., Russ. J. Bioorg. Chem., 2011, vol. 37, no. 2, pp. 198–206.CrossRefGoogle Scholar
  7. 7.
    Yan, S.S., Yan, J., Shi, G., Xu, Q., Chen, S.C., and Tian, Y.W., Expr. Purif., 2005, vol. 40, no. 2, pp. 340–345.CrossRefGoogle Scholar
  8. 8.
    Evans, T.C., Benner, J., and Xu, M.-Q., Protein Sci., 1988, vol. 7, no. 11, pp. 2256–2264.CrossRefGoogle Scholar
  9. 9.
    Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.Q., Gene, 1997, vol. 192, no. 2, pp. 271–281.CrossRefPubMedGoogle Scholar
  10. 10.
    Besset, P.H., Åslund, F., Beckwith, J., and Georgiou, G., Proc. Natl. Acad. Sci. U. S. A., 1999, vol. 96, pp. 13703–13708.CrossRefGoogle Scholar
  11. 11.
    Qiu, J., Swartz, J.R., and Georgiou, G., Appl. Environ. Microbiol., 1998, vol. 64, pp. 4891–4896.PubMedPubMedCentralGoogle Scholar
  12. 12.
    Esipov, R.S. and Kostromina, M.A., Appl. Biochem. Biotechnol., 2015, vol. 175, no. 5, pp. 2468–2488.CrossRefPubMedGoogle Scholar
  13. 13.
    Bradford, M.M., Anal. Biochem., 1976, vol. 72, nos. 1–2, pp. 248–254.CrossRefPubMedGoogle Scholar
  14. 14.
    Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • R. S. Esipov
    • 1
  • V. N. Stepanenko
    • 1
  • I. O. Zvereva
    • 1
  • D. A. Makarov
    • 1
  • M. A. Kostromina
    • 1
  • T. I. Kostromina
    • 1
  • T. I. Muravyova
    • 1
  • A. I. Miroshnikov
    • 1
  • E. V. Grishin
    • 1
  1. 1.Shemyakin–Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia

Personalised recommendations