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

Abstract

Doc number: 82

Abstract

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 μg/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 α (TNF-α) 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 omp F signal sequence. In vitro biological activity evidenced by activation of transcription factor STAT3 and suppression of TNF-α 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.