Optimizing purification and refolding conditions of the recombinant ephrin-A5 from inclusion bodies of Escherichia coli

Authors

  • Alexander V. Zhydzetski Belarusian State University, Niezaliežnasci Avenue, 4, 220030, Minsk, Belarus
  • Mikhail V. Sholukh Belarusian State University, Niezaliežnasci Avenue, 4, 220030, Minsk, Belarus
  • Jürgen Rainer Hannemann Biberach University of Applied Sciences, Karlstrasse Street, 11, 88400, Biberach, Germany
  • Rene Handrick Biberach University of Applied Sciences, Karlstrasse Street, 11, 88400, Biberach, Germany
  • Zhanna V. Motylevich Grodno State Medical University, Horkaha Street, 80, 230009, Grodno, Belarus
  • Juha-Pekk Himanen Memorial Sloan Kettering Cancer Center, 1275 York Avenue, 10065, New York, USA

Keywords:

ephrin-A5, refolding conditions screening, imobilized metal affinity chromatography, recombinant proteins, inclusion bodies
Supporting Agencies
The research was funded by BRFFI within the framework of the treaty No. Б16М-082, program SPSI «Fundamental and applied science – medicine» (assignment 1.11) and international project BMBF (IB-037) «Development of new methods for analyzing specimens of monoclonal antibody glycosilation».

Abstract

The paper is devoted to isolation, purification and development of refolding approach of the recombinant ephrin-A5 out of Escherichia coli inclusion bodies which corresponds to the aim of the research. The main methods applied are UV/visible spectroscopy, protein electrophoresis, column chromatography, refolding by dilution and matrix-assisted renaturation. Composition of the washing and solubilizing solutions was established as a result of washing and solubilizing conditions optimization allowing to get rid of impurities and minimize loss of the target protein as well as carry out the protein extraction out of inclusion bodies. The protein of interest with 70.3 ± 7.4 % purity was managed to obtain in conducting concentration and purification of the solubilized recombinant ephrin-A5 with the help of immobilized metal affinity chromatography on Ni-sepharose. Following refolding system containing 20 mmol/l Tris-HCl; pH 8; 50 mmol/l sucrose; 2.5 mmol/l DTT; 50 mmol/l NaCl with the final protein concentration up to 200 mkg/ml and 10-fold dilution was chosen as the best during the main refolding buffer characteristics screening. 

Author Biographies

  • Alexander V. Zhydzetski, Belarusian State University, Niezaliežnasci Avenue, 4, 220030, Minsk, Belarus

    junior researcher at the research laboratory of the biochemistry department, faculty of biology

  • Mikhail V. Sholukh, Belarusian State University, Niezaliežnasci Avenue, 4, 220030, Minsk, Belarus

    PhD (biology), docent; head of the research laboratory of the biochemistry of metabolism of the biochemistry department, faculty of biology

  • Jürgen Rainer Hannemann, Biberach University of Applied Sciences, Karlstrasse Street, 11, 88400, Biberach, Germany

    PhD (philosophy), full professor; dean of the faculty of biotechnology

  • Rene Handrick, Biberach University of Applied Sciences, Karlstrasse Street, 11, 88400, Biberach, Germany

    PhD (philosophy); head of the research laboratory, faculty of pharmaceutical biotechnology

  • Zhanna V. Motylevich, Grodno State Medical University, Horkaha Street, 80, 230009, Grodno, Belarus

     PhD (biology); head of the international relations department

  • Juha-Pekk Himanen, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, 10065, New York, USA

    doctor of science (philosophy); leading researcher

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Published

2017-12-16

Issue

No. 2 (2017): Journal of the Belarusian State University. Biology

Section

Biochemistry

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How to Cite

Zhydzetski, A. V., Sholukh, M. V., Hannemann, J. R., Handrick, R. ., Motylevich, Z. V., & Himanen, J.-P. . (2017). Optimizing purification and refolding conditions of the recombinant ephrin-A5 from inclusion bodies of Escherichia coli. Experimental Biology and Biotechnology, 2, 58-65. https://journals.bsu.by/index.php/biology/article/view/2446