title: Single-step purification and structural characterization of human interleukin-6 produced in Escherichia 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/fast-atom-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 degrees 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.
authors.affiliation: Dipartimento di Biochimica e Biotecnologie Mediche, II Facoltà di Medicina e Chirurgia, Università di Napoli, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Arcone
authors.forename: P
authors.initials: P
authors.lastname: Pucci
authors.forename: F
authors.initials: F
authors.lastname: Zappacosta
authors.forename: V
authors.initials: V
authors.lastname: Fontaine
authors.forename: A
authors.initials: A
authors.lastname: Malorni
authors.forename: G
authors.initials: G
authors.lastname: Marino
authors.forename: G
authors.initials: G
authors.lastname: Ciliberto
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*
chemical_list: D015850:Interleukin-6; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases