Expression, refolding and purification of a human interleukin-17A variant.
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.
Wu
Bingyuan
B
Biologics Research, Centocor Research and Development Inc., 145 King of Prussia Road, Radnor, PA 19087, USA. bwu63@its.jnj.com
Nemeth
Jennifer F
JF
Janecki
Dariusz J
DJ
Jones
Brian
B
Obmolova
Galina
G
Malia
Thomas J
TJ
Baker
Audrey
A
Bethea
Deidra
D
Elloso
M Merle
MM
Naso
Michael
M
Taudte
Susann
S
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
C508717:IL17A protein, human; D020381:Interleukin-17; D050505:Mutant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins