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




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BMC Biotechnology

, 13:82

Microbial biotechnology

Abstract

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.

KeywordsEscherichia coli Interleukin-10 Outer membrane protein F Inflammatory bowel disease Bacterial transport system AbbreviationsaaAmino acid

EBVEpstein-Barr virus

ELISAEnzyme linked immunosorbent assay

HCMVHuman cytomegalovirus

hIL-10Human interleukin-10

HRPHorseradish peroxidase

IBDInflammatory bowel disease

IL-10Interleukin-10

IL-10RInterleukin-10 receptor

LPSLipopolysaccharide

ODOptical density

OmpAOuter membrane protein A

OmpCOuter membrane protein C

OmpFOuter membrane protein F

PCRPolymerase chain reaction

PelBPectate lyase B

PhoAAlkaline phosphatase

STAT3Signal transducer and activator of transcription 3

StIIHeat-stable enterotoxin 2

TMBTetramethylbenzidine

TNF-αTumor necrosis factor alpha

TyrTyrosine

vIL-10Viral interleukin-10.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-13-82 contains supplementary material, which is available to authorized users.

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Author: Sarah Förster - Manuela Brandt - Dorothea S Mottok - Anke Zschüttig - Kurt Zimmermann - Frederick R Blattner - Florian 

Source: https://link.springer.com/







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