title: Expression, refolding and purification of a human interleukin-17A variant.
abstract: A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.
authors.affiliation: Biologics Research, Centocor Research and Development Inc., 145 King of Prussia Road, Radnor, PA 19087, USA. bwu63@its.jnj.com
authors.forename: Bingyuan
authors.initials: B
authors.lastname: Wu
authors.forename: Jennifer F
authors.initials: JF
authors.lastname: Nemeth
authors.forename: Dariusz J
authors.initials: DJ
authors.lastname: Janecki
authors.forename: Brian
authors.initials: B
authors.lastname: Jones
authors.forename: Galina
authors.initials: G
authors.lastname: Obmolova
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Malia
authors.forename: Audrey
authors.initials: A
authors.lastname: Baker
authors.forename: Deidra
authors.initials: D
authors.lastname: Bethea
authors.forename: M Merle
authors.initials: MM
authors.lastname: Elloso
authors.forename: Michael
authors.initials: M
authors.lastname: Naso
authors.forename: Susann
authors.initials: S
authors.lastname: Taudte
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D020381:Interleukin-17 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: C508717:IL17A protein, human; D020381:Interleukin-17; D050505:Mutant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins