Biotechnology and Applied Biochemistry 2014-01-01

Matrix-assisted refolding and purification of placenta-derived recombinant human interleukin-6 produced in Escherichia coli.

Nadeem Ahmed, Ahmad Usman Zafar, Mohsin Ahmad Khan, Saad Tahir, Muhammad Islam Khan, Hamid Bashir, Faidad Khan, Samreen Sarwar, Sadaf Ilyas, Tayyab Husnain

Index: Biotechnol. Appl. Biochem. 61(5) , 541-8, (2014)

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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. © 2014 International Union of Biochemistry and Molecular Biology, Inc.