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Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2)

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Abstract

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1–155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.

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Abbreviations

ECS:

embryonic calf serum

FGF-2:

basic fibroblast growth factor

FGFR:

receptor of fibroblast growth factor

IPTG:

isopropyl-β-D-1-thiogalactopyranoside

MTT:

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide

TB:

Terrific Broth (enriched medium)

Trx:

thioredoxin

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Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    M. E. Gasparian, P. A. Elistratov, D. A. Dolgikh & M. P. Kirpichnikov

  2. National Hematological Scientific Center, Russian Academy of Medical Sciences, Novozykovsky pr. 4a, 125167, Moscow, Russia

    N. I. Drize & I. N. Nifontova

Authors
  1. M. E. Gasparian
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  2. P. A. Elistratov
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  3. N. I. Drize
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  4. I. N. Nifontova
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  5. D. A. Dolgikh
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  6. M. P. Kirpichnikov
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Correspondence to M. E. Gasparian.

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Original Russian Text © M. E. Gasparian, P. A. Elistratov, N. I. Drize, I. N. Nifontova, D. A. Dolgikh, M. P. Kirpichnikov, 2009, published in Biokhimiya, 2009, Vol. 74, No. 2, pp. 272–277.

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Gasparian, M.E., Elistratov, P.A., Drize, N.I. et al. Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2). Biochemistry Moscow 74, 221–225 (2009). https://doi.org/10.1134/S000629790902014X

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  • DOI: https://doi.org/10.1134/S000629790902014X

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