title: A rapid, two-step method for high-yield purification of recombinant rat acidic and basic fibroblast growth factors.
abstract: We describe the preparation of vectors for T7 polymerase-driven, high-level expression of rat acidic (aFGF) or basic (bFGF) fibroblast growth factors in Escherichia coli. Following isopropyl-beta-D-thiogalactoside induction of T7 polymerase, rat aFGF or bFGF represented a major portion of the proteins synthesized by vector-transformed E. coli. Passage of cell extracts through an Amicon YM-100 membrane provided ultrafiltrates containing either aFGF or bFGF as the principal component. A single heparin-Sepharose chromatography of the ultrafiltrates yielded essentially homogenous, biologically active, recombinant rat aFGF or bFGF. By silanizing vessels and using buffers containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, we could store homogenous aFGF or bFGF preparations without significant loss during repeated freezing and thawing. Previous methods for purifying aFGF or bFGF utilized salt fractionation followed by sequential ion exchange and heparin-Sepharose chromatography. These purified aFGF or bFGF preparations routinely were stored in buffered carrier protein to minimize mitogen loss resulting from adsorption to glass or plastic surfaces. In contrast, the methods that we detail are rapid, require minimal manipulation of preparations, and permit storage of carrier-free, homogenous preparations without loss resulting from surface adsorption. The protocols can be used for preparation of homogenous aFGF or bFGF of other species and may be readily applied to the isolation and characterization of FGF-like mitogens present in cultured cell extracts, conditioned medium, or tissue preparations.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002455:Cell Division / Q000187:drug effects; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D016220:Fibroblast Growth Factor 1 / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects; D006493:Heparin; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D051381:Rats / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012685:Sepharose; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D007544:Isopropyl Thiogalactoside; D006493:Heparin; D012685:Sepharose; D001616:beta-Galactosidase