Single‐step purification and structural characterization of human interleukin‐6 produced in Esherichia coli From a T7 RNA polymerase expression vector

Abstract

Human interleukin‐6 or B‐cell stimulatory factor‐2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin‐6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin‐6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin‐6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic‐exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high‐pressure liquid chromatography/fastatom‐bombardment mass spectrometry demonstrates that recombinant human interleukin‐6 is identical to the natural protein both in amino acid sequence and S‐S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met‐Ala dipeptide extension at the N‐terminus. Purified recombinant human interleukin‐6 is biologically active because it is able to induce at least 70‐fold the human C‐reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4°C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin‐6 (about 25 mg/l) combined with a very simple purification scheme. Copyright © 1991, Wiley Blackwell. All rights reserved