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

BMC Biotechnology

Research Article

Sarah Förster¹  Florian Gunzer¹  Manuela Brandt¹  Anke Zschüttig¹  Christoph Pöhlmann²  Dorothea S Mottok³  Frederick R Blattner⁴  Kurt Zimmermann⁵ 

[1] Institute of Medical Microbiology and Hygiene, TU Dresden, Fiedlerstrasse 42, 01307, Dresden, Germany;Institute of Medical Microbiology and Hygiene, TU Dresden, Fiedlerstrasse 42, 01307, Dresden, Germany;Department of Laboratory Medicine, Robert-Bosch Hospital, Auerbachstrasse 110, 70376, Stuttgart, Germany;Institute of Medical Microbiology and Hygiene, TU Dresden, Fiedlerstrasse 42, 01307, Dresden, Germany;QIAGEN Hamburg GmbH, Königstrasse 4a, 22767, Hamburg, Germany;Scarab Genomics LLC, 1202 Ann St., 53713, Madison, Wisconsin, USA;Department of Genetics, University of Wisconsin, 425G Henry Mall, 53706-1580, Madison, Wisconsin, USA;SymbioPharm GmbH, Auf den Lüppen 8, 35745, Herborn-Hörbach, Germany;

关键词: Escherichia coli;    Interleukin-10;    Outer membrane protein F;    Inflammatory bowel disease;    Bacterial transport system;   

DOI  :  10.1186/1472-6750-13-82

 received in 2012-11-23, accepted in 2013-09-26,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundInterleukin-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.ResultsThe 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.ConclusionsIn 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-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.

【 授权许可】

Unknown   
© Förster et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.