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Optimization of the refolding of recombinant human granulocyte-macrophage colony-stimulating factor immobilized on affinity sorbent

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Abstract

A method for the production and purification of biologically active recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), expressed in Escherichia coli, has been developed. Washing of an inclusion body fraction which allows the removal of numerous ballast proteins and on-column refolding of rhGM-CSF are specific characteristics of the method. The refolding efficiency reached 70%; the purity of the preparation constituted 95%. The biological activity was similar to those of other recombinant hGM-CSF analogs.

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Abbreviations

APC:

antigen-presenting cells

GM-CSF:

granulocyte-macrophage colony-stimulating factor

DMSO:

dimethylsulphoxide

IEA:

immune enzyme assay

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OD:

optical density

PAAG:

polyacrylamide gel

PMSF:

phenilmethylsulfonyl fluoride

PCR:

polymerase chain reaction

rhGM:

CSF-human recombinant granulocyte-macrophage colony-stimulating factor

LB medium:

Luria-Bertani medium

IBs:

inclusion bodies

TNF:

tumor necrosis factor

EMEA:

European Agency for the Evaluation of Medicinal Products

IPTG:

isopropyl [beta]-D-1-thiogalactopyranoside

FDA:

Food and Drug Administration, United States

HEPES:

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

G-CSF:

granulocyte colony stimulating factor

M-CSF:

macrophage colony-stimulating factor

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Authors and Affiliations

  1. Federal State Budgetary Institution “N.N. Blokhin Russian Cancer Research Center” under Russian Academy of Medical Sciences, Russian Academy of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478, Russia

    E. N. Kosobokova, K. A. Skrypnik, M. V. Pinyugina, A. I. Shcherbakov & V. S. Kosorukov

Authors
  1. E. N. Kosobokova
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  2. K. A. Skrypnik
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  3. M. V. Pinyugina
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  4. A. I. Shcherbakov
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  5. V. S. Kosorukov
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Correspondence to E. N. Kosobokova.

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Original Russian Text © E.N. Kosobokova, K.A. Skrypnik, M.V. Pinyugina, A.I. Shcherbakov, V.S. Kosorukov, 2013, published in Biotekhnologiya, 2013, No. 3, pp. 39–46.

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Kosobokova, E.N., Skrypnik, K.A., Pinyugina, M.V. et al. Optimization of the refolding of recombinant human granulocyte-macrophage colony-stimulating factor immobilized on affinity sorbent. Appl Biochem Microbiol 50, 773–779 (2014). https://doi.org/10.1134/S0003683814080031

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  • DOI: https://doi.org/10.1134/S0003683814080031

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