Production of recombinant IGF1 and its action on neuroblastoma cells in vitro

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  • Authors: Ishuk S.A.1, Bogomolova E.G.1,2, Dobrovolskaya O.A.1, Akhmetshina A.O.2, Krasnoshchek D.S.3, Lukovenko A.A.2, Fedorova E.A.1, Klyus A.M.4, Kolmakov N.N.1, Zherebtsova J.V.1, Dukhovlinov I.V.1, Klimov N.A.1, Simbirtsev A.S.1
  • Affiliations:
    1. Institute of Experimental Medicine
    2. Limited Liability Company “Biochemical Agent”
    3. Pavlov First Saint Petersburg State Medical University
    4. Limited Liability Company “Infarm Consulting”
  • Issue: Vol 18, No 4 (2018)
  • Pages: 34-41
  • Section: Articles
  • URL: https://journals.eco-vector.com/MAJ/article/view/11683
  • DOI: https://doi.org/10.17816/MAJ18434-41
  • Cite item
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Abstract


This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein.

To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography.

The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons.

Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.


Sergey A. Ishuk

Institute of Experimental Medicine

Author for correspondence.
Email: s.ischuk.spb@gmail.com

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

Postgraduate Student, Department of Medical Biotechnology and Immunopharmacology

Elena G. Bogomolova

Institute of Experimental Medicine; Limited Liability Company “Biochemical Agent”

Email: bogomolovaele@inbox.ru

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376; Saint-Petersburg

Postgraduate Student, Department of Medical Biotechnology and Immunopharmacology; Deputy Director for Science

Olga A. Dobrovolskaya

Institute of Experimental Medicine

Email: dobrovolskaya-oly@yandex.ru

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

Research Worker, Department of Medical Biotechnology and Immunopharmacology

Alyona O. Akhmetshina

Limited Liability Company “Biochemical Agent”

Email: akhmetshinaalena@gmail.com

Russian Federation, Saint Petersburg

Research Worker

Daria S. Krasnoshchek

Pavlov First Saint Petersburg State Medical University

Email: dkrasnoshchek@list.ru

Russian Federation, 6/8, Lva Tolstogo street, St. Petersburg, 197089

6th year student

Anna A. Lukovenko

Limited Liability Company “Biochemical Agent”

Email: a.lukovenko@yahoo.com

Russian Federation, Saint Petersburg

Junior research assistant

Ekaterina A. Fedorova

Institute of Experimental Medicine

Email: science.eaf@yandex.ru

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

Research Worker, Department of Medical Biotechnology and Immunopharmacology

Alexander M. Klyus

Limited Liability Company “Infarm Consulting”

Email: Inffarmcon@gmail.com

Russian Federation, Saint Petersburg

Director General

Nikolay N. Kolmakov

Institute of Experimental Medicine

Email: ashvin.nick@gmail.com

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

Research Worker, Department of Molecular Genetics

Julia V. Zherebtsova

Institute of Experimental Medicine

Email: juliazh@yandex.ru

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

Junior research assistant, Department of Medical Biotechnology and Immunopharmacology

Ilya V. Dukhovlinov

Institute of Experimental Medicine

Email: dukhovlinov@gmail.com

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

PhD in Biology, Head of the Laboratory of Protein Genetic Engineering, Department of Medical Biotechnology and Immunopharmacology

Nikolai A. Klimov

Institute of Experimental Medicine

Email: nklimov@mail.ru

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

MD, PhD, Leading research associate, Department of Molecular Biotechnology and Immunopharmacology

Andrey S. Simbirtsev

Institute of Experimental Medicine

Email: simbas@mail.ru

Russian Federation, 12, Academic Pavlov street, Saint-Petersburg, 197376

PhD in Biology, corresponding member Russian Academy of Sciences, Head of the Department of Medical Biotechnology and Immunopharmacology

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Supplementary files

Supplementary Files Action
1. Fig. 1. Electrophoregram of proteins obtained from lysates of E. coli BL21 cells (pET28-IGF-1) after induction of the synthesis of recombinant human IGF-1: 1 — Thermo Fisher Scientific molecular weight marker; 2 - control (night culture without inductor); 3 - induction of 1 mM IPTG, 5 hours; 4 - induction of 0.2% lactose by the method of Studier, 18 hours View (155KB) Indexing metadata
2. Fig. 2. Purification of recombinant human IGF-1 by metal chelate chromatography. 80 mg of protein is applied. On the chromatogram: line [1] - optical density at a wavelength of 280 nm; line [2] - imidazole gradient View (130KB) Indexing metadata
3. Fig. 3. Electrophoresis in 12.5% PAGE of purified recombinant human IGF-1: 1 — Thermo Fisher Scientific molecular weight markers; 2 - 5 µg of recombinant protein IGF-1, purified on a SP-Sepharose column View (54KB) Indexing metadata
4. Fig. 4. The effect of recombinant IGF-1 on the differentiation of Neuro 2a cells in vitro: a - Neuro 2a cells grown in serum-free medium for 48 hours (× 100); b - Neuro 2a cells grown similarly to (a) with the addition of 100 ng / ml IGF-1 (× 100); c - Neuro 2a cells grown in serum-free medium for 3 hours, followed by return to growth medium (positive control of differentiation) (× 100) View (451KB) Indexing metadata

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Copyright (c) 2018 Ishuk S.A., Bogomolova E.G., Dobrovolskaya O.A., Akhmetshina A.O., Krasnoshchek D.S., Lukovenko A.A., Fedorova E.A., Klyus A.M., Kolmakov N.N., Zherebtsova Y.V., Dukhovlinov I.V., Klimov N.A., Simbirtsev A.S.

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