title: Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal affinity chromatography.
abstract: The construction and use of a plasmid which allows the expression and single step purification of recombinant rat basic fibroblast growth factor (bFGF) is described. A cDNA encoding rat bFGF was subcloned into the expression plasmid pQE-9 (Qiagen) in such a way that the bFGF which is produced from the resulting construct contains 6 histidine residues near the amino terminus. The resulting plasmid, pQE-9-bFGF, was expressed in the E. coli strain M15[pREP4] and the 6 x His-bFGF was purified to homogeneity from the soluble fraction of the bacterial cell lysate in a single step by affinity chromatography on a nickel chelate resin. About 5 mg of 6 x His-bFGF was obtained from the soluble fraction from one liter of bacterial cell culture. Testing of the 6 x His-bFGF in a PC12 cell differentiation assay showed that its activity was comparable to the activities for native bFGF and recombinant bFGF purified by multistep methods.
authors.affiliation: Department of Biological Chemistry, University of California, Irvine 92717-1700, USA.
authors.forename: M
authors.initials: M
authors.lastname: Kroiher
authors.forename: S
authors.initials: S
authors.lastname: Raffioni
authors.forename: R E
authors.initials: RE
authors.lastname: Steele
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D006639:Histidine; D008969:Molecular Sequence Data; D009532:Nickel; D016716:PC12 Cells / Q000187:drug effects; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D006639:Histidine; D009532:Nickel