title: Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2).
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.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. marine_gasparian@yahoo.com
authors.forename: M E
authors.initials: ME
authors.lastname: Gasparian
authors.forename: P A
authors.initials: PA
authors.lastname: Elistratov
authors.forename: N I
authors.initials: NI
authors.lastname: Drize
authors.forename: I N
authors.initials: IN
authors.lastname: Nifontova
authors.forename: D A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009154:Mutation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2