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Overexpression in Escherichia coli and Purification of Human Fibroblast Growth Factor (FGF-2)

M. E. Gasparian1*, P. A. Elistratov1, N. I. Drize2, I. N. Nifontova2, D. A. Dolgikh1, and M. P. Kirpichnikov1

1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 335-2888; E-mail: marine_gasparian@yahoo.com

2National Hematological Scientific Center, Russian Academy of Medical Sciences, Novozykovsky pr. 4a, 125167 Moscow, Russia; fax: (495) 614-9269; E-mail: iranifontova@yandex.ru

* To whom correspondence should be addressed.

Received April 17, 2008; Revision received May 26, 2008
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

DOI: 10.1134/S000629790902014X