title: Periplasmic delivery of biologically active human interleukin-10 in Escherichia coli via a sec-dependent signal peptide.
abstract: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10.
authors.affiliation: Institute of Medical Microbiology and Hygiene, Dresden University of Technology, Dresden, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Pöhlmann
authors.forename: Manuela
authors.initials: M
authors.lastname: Brandt
authors.forename: Dorothea S
authors.initials: DS
authors.lastname: Mottok
authors.forename: Anke
authors.initials: A
authors.lastname: Zschüttig
authors.forename: John W
authors.initials: JW
authors.lastname: Campbell
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Blattner
authors.forename: David
authors.initials: D
authors.lastname: Frisch
authors.forename: Florian
authors.initials: F
authors.lastname: Gunzer
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D015870:Gene Expression; D016753:Interleukin-10 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D019897:Periplasm / Q000378:metabolism*; D055503:Protein Multimerization; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; C508609:IL10 protein, human; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10