title: Matrix-assisted refolding and purification of placenta-derived recombinant human interleukin-6 produced in Escherichia coli.
abstract: Biological activity of human interleukin-6 (IL-6) is associated with a vast number of diseases such as rheumatoid arthritis, sepsis, and severe inflammatory diseases. In this study, human IL-6 cDNA was isolated from a cDNA library that was constructed with mRNA derived from human placental tissues. Subsequently, the complete human IL-6 cDNA was cloned and expressed in BL21DE3 cells. The recombinant human IL-6 (rhIL-6) protein was expressed in a form of an insoluble inclusion body. Inclusion bodies were solubilized under denaturing conditions and purified by immobilized metal affinity chromatography with gradual on-column refolding by the gradient elution method (from 6 to 0 M urea). The protein was purified to apparent homogeneity of about 99% with a yield of 50 mg/L. The purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high-performance liquid chromatography, and Western blotting analysis. The bioactivity was assessed by proliferation assay of TF-1 cells in a dose-dependent manner. The present study confirms the expression of the placenta-derived IL-6 gene in a prokaryotic expression system and matrix-assisted on-column refolding and purification of rhIL-6 by immobilized metal affinity chromatography.
authors.affiliation: National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
authors.forename: Nadeem
authors.initials: N
authors.lastname: Ahmed
authors.forename: Ahmad Usman
authors.initials: AU
authors.lastname: Zafar
authors.forename: Mohsin Ahmad
authors.initials: MA
authors.lastname: Khan
authors.forename: Saad
authors.initials: S
authors.lastname: Tahir
authors.forename: Muhammad Islam
authors.initials: MI
authors.lastname: Khan
authors.forename: Hamid
authors.initials: H
authors.lastname: Bashir
authors.forename: Faidad
authors.initials: F
authors.lastname: Khan
authors.forename: Samreen
authors.initials: S
authors.lastname: Sarwar
authors.forename: Sadaf
authors.initials: S
authors.lastname: Ilyas
authors.forename: Tayyab
authors.initials: T
authors.lastname: Husnain
mesh_terms: D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006639:Histidine; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004247:DNA
keywords: chelating sepharose fast flow; histidine tags; immobilized metal-ion-affinity chromatography; on-column refolding; pET28 expression vector; recombinant proteins