title: Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli.
abstract: Wild-type human interleukin-10 (hIL-10) is a non-covalent homodimer with a short half-life, thus limiting its therapeutic applications in vivo. To avoid loss of function due to dimer dissociation, we designed a synthetic hIL-10 analog by bridging both monomers via a 15 amino acid-long peptide spacer in a C-terminal to N-terminal fashion. For secretory expression in Escherichia coli, a 1156 bp fragment was generated from template vector pAZ1 by fusion PCR encoding a T7 promoter region and the signal sequence of the E. coli outer membrane protein F fused in frame to two tandem E. coli codon-optimized mature hIL-10 genes connected via a 45 nucleotide linker sequence. The construct was cloned into pUC19 for high-level expression in E. coli BL21 (DE3). The mean concentrations of hIL-10 fusion protein in the periplasm and supernatant of E. coli at 37 °C growth temperature were 130 ± 40 and 2 ± 1 ng/ml, respectively. The molecular mass of the recombinant protein was assessed via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, indicating correct processing of the signaling sequence in E. coli. In vitro biological activity was shown by phosphorylation of signal transducer and activator of transcription protein 3 and suppression of tumor necrosis factor α secretion in lipopolysaccharide-stimulated macrophages.
authors.affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Fetscherstrasse 74, 01307, Dresden, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Guggenbichler
authors.affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Fetscherstrasse 74, 01307, Dresden, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Büttner
authors.affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Fetscherstrasse 74, 01307, Dresden, Germany.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Rudolph
authors.affiliation: SymbioPharm GmbH, Auf den Lüppen 8, 35745, Herborn-Hörbach, Germany.
authors.forename: Kurt
authors.initials: K
authors.lastname: Zimmermann
authors.affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Fetscherstrasse 74, 01307, Dresden, Germany. florian.gunzer@tu-dresden.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Gunzer
authors.affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Fetscherstrasse 74, 01307, Dresden, Germany. christoph.poehlmann@klinikum-memmingen.de.|Department of Medical Microbiology and Hygiene, Hospital of Memmingen, Bismarckstrasse 23, 87700, Memmingen, Germany. christoph.poehlmann@klinikum-memmingen.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Pöhlmann
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015870:Gene Expression; D016753:Interleukin-10 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C508609:IL10 protein, human; D011993:Recombinant Fusion Proteins; D016753:Interleukin-10
keywords: Bacterial transport system; Escherichia coli; Inflammatory bowel disease; Interleukin-10; Outer membrane protein F