High-Level Expression of Human BSF-2/IL-6 cDNA in Escherichia coli Using a New Type of Expression-Preparation System

Abstract

BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.

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

“…Glycosylation, however, is not essential for IL-6 activity because the protein produced in Escherichiu coli shows the same specific activity as natural IL-6 in biological assays on B-cells, hybridomas [6] and hepatomas in culture (this study).…”

“…Two different systems have been described for the overproduction of IL-6 in E. coli [6,71. In both cases, in order to increase stability in bacterial cells, the cytokine has been synthesized as a fusion protein either with the human interleukin-2 N-terminal peptide [6] or with the human growth hormone [7].…”

“…In both cases, in order to increase stability in bacterial cells, the cytokine has been synthesized as a fusion protein either with the human interleukin-2 N-terminal peptide [6] or with the human growth hormone [7]. These fusion proteins have to be subjected to cleavage in vitro by specific endopeptidases and several purification steps are required in order to obtain the pure protein.…”

Interleukin‐6: Structure‐function relationships

“…IL-6 has been expressed in rabbit reticulocyte lysate (Geiger et al, 1988), Escherichia coli (Brakenhoff et al, 1987;Tonouchi et al, 1988;Kriittgen et al, 1990a;Yasueda et al, 1990;Arcone et al, 1991a;Grennet et al, 1991;Zhang et al, 1992;Hammacher et al, 1994), Saccharomyces cerevisiae (Guisez et al, 1991), Pichia pastoris (this laboratory, unpublished data), Aspergillus nidulans , Xenopus laevis (Fontaine et al, 1991), baculovirus insect cells and Chinese hamster ovary (CHO) cells (Eisenthal et al, 1993;Orita et al, 1994).…”

Tyrosine 759 of the cytokine receptor gp130 is involved in Listeria monocytogenes susceptibility

“…Peritoneal macrophages were cultured overnight at 37°C, 5% CO 2 in the complete medium with or without LPS (E. coli O55:B5, Sigma), IFN␥ (Peprotech EC Ltd, London, UK) and recombinant human IL-6. 30 The NO − 2 concentration in the culture supernatant was measured with Griess reagent.…”