Production and purification of refolded recombinant human IL-7 from inclusion bodies.
A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.
Ouellette
Thomas
T
Biopharmaceutical Development Program, National Cancer Institute at Frederick, SAIC-Frederick, Inc., Frederick, MD 21702-1201, USA.
Destrau
Sophie
S
Ouellette
Timothy
T
Zhu
Jianwei
J
Roach
John M
JM
Coffman
J Daniel
JD
Hecht
Toby
T
Lynch
James E
JE
Giardina
Steven L
SL
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005285:Fermentation; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D015851:Interleukin-7 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D015851:Interleukin-7; D011994:Recombinant Proteins