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Biochemistry (Moscow)

, Volume 74, Issue 2, pp 221–225 | Cite as

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

  • M. E. GasparianEmail author
  • P. A. Elistratov
  • N. I. Drize
  • I. N. Nifontova
  • D. A. Dolgikh
  • M. P. Kirpichnikov
Article

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.

Key words

growth factors FGF-2 recombinant proteins E. coli 

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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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • M. E. Gasparian
    • 1
    Email author
  • P. A. Elistratov
    • 1
  • N. I. Drize
    • 2
  • I. N. Nifontova
    • 2
  • D. A. Dolgikh
    • 1
  • M. P. Kirpichnikov
    • 1
  1. 1.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.National Hematological Scientific CenterRussian Academy of Medical SciencesMoscowRussia

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