Production and Purification of Refolded Recombinant Human IL-7 From Inclusion Bodies
Abstract
A recombinant clone of human rhIL-72 that codes for 152 amino acids was cloned into Escherichia coli HMS 174(DE3) pLys S under the control of a T7 promoter. The cloned rhIL-7 has a molecular weight of 17.4 kDa with three disulfide bonds and three sites for glycosylation. The molecule has a hydrophobic nature and heparin affinity. The isoelectric point of this recombinant protein is 9.3 by CZE and 8.45 by AAA. The insoluble inclusion bodies produced during fermentation were solubilized by homogenization with 6M guanidine HCl. The rhIL-7 inclusion bodies were enriched for monomer by size exclusion chromatography using a Superdex 200 Prep Grade solumn under denaturing conditions. The enriched, denatured rhIL-7 monomer was reduced and stratically refolded at a final protein concentration of 80 - 100 g/ml and a final guanidine HCl concentration of 0.06 M at 2-8 C for 48 - 72 hours. The refolded monmeric rhIL-7 was subsequently purified by low pressure liquid chromatography using hydrophobic interaction cation exchange and size exclusion chromatography. The purified final product was >95% pure by Coomassie brilliant blue -stained SDS-PAGE, high pressure size exclusion chromatography 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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2003
- Accession Number
- ADA430155
Entities
People
- Jianwei Zhu
- John M. Roach
- Sophie Destrau
- Thomas Ouelette
- Timothy Ouelette
Organizations
- United States Army Medical Research Institute of Infectious Diseases