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

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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.

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About the authors

Sergey A. Ishuk

Institute of Experimental Medicine

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

Postgraduate Student, Department of Medical Biotechnology and Immunopharmacology

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

Elena G. Bogomolova

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

Email: bogomolovaele@inbox.ru

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

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

Olga A. Dobrovolskaya

Institute of Experimental Medicine

Email: dobrovolskaya-oly@yandex.ru

Research Worker, Department of Medical Biotechnology and Immunopharmacology

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

Alyona O. Akhmetshina

Limited Liability Company “Biochemical Agent”

Email: akhmetshinaalena@gmail.com

Research Worker

Russian Federation, Saint Petersburg

Daria S. Krasnoshchek

Pavlov First Saint Petersburg State Medical University

Email: dkrasnoshchek@list.ru

6th year student

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

Anna A. Lukovenko

Limited Liability Company “Biochemical Agent”

Email: a.lukovenko@yahoo.com

Junior research assistant

Russian Federation, Saint Petersburg

Ekaterina A. Fedorova

Institute of Experimental Medicine

Email: science.eaf@yandex.ru

Research Worker, Department of Medical Biotechnology and Immunopharmacology

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

Alexander M. Klyus

Limited Liability Company “Infarm Consulting”

Email: Inffarmcon@gmail.com

Director General

Russian Federation, Saint Petersburg

Nikolay N. Kolmakov

Institute of Experimental Medicine

Email: ashvin.nick@gmail.com

Research Worker, Department of Molecular Genetics

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

Julia V. Zherebtsova

Institute of Experimental Medicine

Email: juliazh@yandex.ru

Junior research assistant, Department of Medical Biotechnology and Immunopharmacology

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

Ilya V. Dukhovlinov

Institute of Experimental Medicine

Email: dukhovlinov@gmail.com

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

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

Nikolai A. Klimov

Institute of Experimental Medicine

Email: nklimov@mail.ru

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

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

Andrey S. Simbirtsev

Institute of Experimental Medicine

Email: simbas@mail.ru

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

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

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2. 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

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3. 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

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4. 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

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5. 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)

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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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