Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct

BMC Biotechnology Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct Sarah Frster 0 Manuela Brandt 0 Dorothea S Mottok 0 Anke Zschttig 0 Kurt Zimmermann Frederick R Blattner Florian Gunzer 0 Christoph Phlmann 0 1 0 Institute of Medical Microbiology and Hygiene, TU Dresden , Fiedlerstrasse 42, 01307 Dresden , Germany 1 Department of Laboratory Medicine, Robert-Bosch Hospital , Auerbachstrasse 110, 70376 Stuttgart , Germany Background: Interleukin-10 homologues encoded by Herpes viruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) hold interesting structural and biological characteristics compared to human interleukin-10 (hIL-10) that render these proteins promising candidates for therapeutic application in inflammatory bowel disease (IBD). Intestinal delivery of cytokines using bacterial carriers as chassis represents a novel approach for treatment of IBD patients. For proof of concept, a Sec-dependent transporter construct was designed for secretory expression of recombinant viral IL-10 proteins in the periplasm of Escherichia coli laboratory strain BL21 (DE3), which might serve as part of a prospective lysis based delivery and containment system. Results: The signal peptide of E. coli outer membrane protein F fused to the mature form of the viral IL-10 proteins enabled successful transport into the periplasm, a compartment which seems crucial for proper assembly of the dimeric configuration of the cytokines. Cytokine concentrations in different bacterial compartments were determined by ELISA and achieved yields of 67.8 ng/ml 24.9 ng/ml for HCMV IL-10 and 1.5 g/ml 841.4 ng/ml for EBV IL-10 in the periplasm. Immunoblot analysis was used to confirm the correct size of the E. coli-derived recombinant cytokines. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the signal transduction cascade after IL-10 receptor interaction, as well as suppression of tumor necrosis factor (TNF-) release of lipopolysaccharide-stimulated mouse macrophages were used as read-out assays for proving in vitro biological activity of the E. coli derived, recombinant viral IL-10 counterparts. Conclusions: In this study, proof of principle is provided that E. coli cells are a suitable chassis for secretory expression of viral IL-10 cytokines encoded by codon-optimized synthetic genes fused to the E. coli ompF signal sequence. In vitro biological activity evidenced by activation of transcription factor STAT3 and suppression of TNF- in mammalian cell lines was shown to be strictly dependent on export of viral IL-10 proteins into the periplasmic compartment. E. coli might serve as carrier system for in situ delivery of therapeutic molecules in the gut, thus representing a further step in the development of novel approaches for treatment of IBD. Escherichia coli; Interleukin-10; Outer membrane protein F; Inflammatory bowel disease; Bacterial transport system - Background Inflammatory bowel disease (IBD), such as Colitis ulcerosa and Crohns disease, present with symptoms like diarrhoea, abdominal pain and weight loss which often means a long ordeal for the patients as well as a great effort for the health care system. Though wide parts of the underlying pathogenesis are still unknown, main components have been described and open up the opportunity for novel therapeutic approaches. Profound downregulation of the auto-inflammatory process has recently been attempted with antibodies against tumor necrosis factor (TNF-) [1]. A totally new conception is the in situ delivery of the anti-inflammatory cytokine interleukin-10 (IL-10) via bacterial carrier systems. Steidler et al. showed decreased inflammation in chemically induced colitis of mice treated with a Lactococcus lactis strain secreting murine IL-10 [2]. Since human IL-10 (hIL-10) possesses not only antiinflammatory properties like down-regulation of proinflammatory cytokines, inhibition of antigen presentation on dendritic cells or suppression of major histocompatibility complex expression, but also displays pro-inflammatory activity such as stimulation of B-cell maturation and proliferation of natural killer cells [3], IL-10 homologues encoded by members of the Herpes virus family move into the focus of interest. Human cytomegalo- (HCMV) and Epstein-Barr virus (EBV) perfected their strategies to avoid eradication by the immune system during co-evolution with the host [4]. The EBV and HCMV IL-10 counterparts encoded by the BCRF1 and UL111A gene region, respectively, enable Herpes viruses among other mechanisms to escape the hosts immune system and to establish a latent, lifelong infection. Viral IL-10 homologues share many biological activities of hIL-10 but, due to selective pressure during virus evolution, also display unique traits such as increased molecule stability and lack of immunostimulatory functions [5-7]. These characteristics suggest the viral counterparts to be even more effective than hIL-10 as immunosuppressants. Thus, recombinant viral IL-10 (vIL-10) proteins emerge as promising candidates for therapeutic applications. So far, only EBV IL-10 has been successfully expressed in both, prokaryotic and eukaryotic expression systems, which, however, required further steps to yield a functional protein [8,9]. We aim at using Escherichia coli as chassis for intestinal delivery of recombinant vIL-10 proteins in IBD patients. In a recent study, we have demonstrated that the bacterial periplasm is a suitable milieu for expression of biologically active recombinant IL-10 [10]. An inducible cell lysis device may then confer both, biological containment and release of IL-10 into the culture medium [11]. Thus, as proof of concept, a Sec-dependent vIL-10 transporter was constructed in laboratory E. coli strain BL21 (DE3) which allows secretory expression of codon optimized viral IL-10 genes in the E. coli periplasm. Translocation of recombinant viral proteins into E. coli periplasm was achieved by fusing the signal peptide of the E. coli outer membrane protein F (OmpF) to the mature form of the vIL-10 proteins. The biological activity of the recombinant viral proteins was proved by two independent cell-based in vitro assays. To our knowledge, we describe here for the first time the successful secretory expression of biologically active viral IL-10 homologues in a prokaryotic chassis without further purification steps. Results and discussion Design and cloning of the artificial vIL-10 transporters An E. coli codon optimized nucleotide sequence was generated from the viral IL-10 gene sequences (HCMV IL-10: 477 bp, GenBank accession number 1LQS_M; EBVIL10: 441 bp, GenBank accession number YP_401634). The original signal sequences of the viral IL-10 genes were replaced by the first 66 nucleotides of the ompF gene of E. coli K12-MG1655 (GenBank accession number NC_000913.2) (...truncated)