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Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli

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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.

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Acknowledgments

This work was funded by a grant of the “MeDDrive38” program of the TU Dresden, Medical Faculty, Carl Gustav Carus to C.P. and by a grant of the Federal Ministry of Education and Research (BMBF Genomik-Transfer, PROTumor consortium, project number 0315590A) to F.G. We would like to thank Manuela Brandt for excellent technical assistance and Christian Auerbach and Claudia Dittrich for help with database submission of plasmid sequences and bacterial strains, and we would also like to acknowledge Dr. Michael Hogardt (Max von Pettenkofer Institute, Munich, Germany) for providing the murine macrophage cell line J-774.1.

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Authors and Affiliations

  1. Institute of Medical Microbiology and Hygiene, TU Dresden, Fetscherstrasse 74, 01307, Dresden, Germany

    Florian Guggenbichler, Carolin Büttner, Wolfram Rudolph, Florian Gunzer & Christoph Pöhlmann

  2. SymbioPharm GmbH, Auf den Lüppen 8, 35745, Herborn-Hörbach, Germany

    Kurt Zimmermann

  3. Department of Medical Microbiology and Hygiene, Hospital of Memmingen, Bismarckstrasse 23, 87700, Memmingen, Germany

    Christoph Pöhlmann

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  1. Florian Guggenbichler
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  2. Carolin Büttner
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  3. Wolfram Rudolph
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  4. Kurt Zimmermann
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  5. Florian Gunzer
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  6. Christoph Pöhlmann
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Correspondence to Florian Gunzer or Christoph Pöhlmann.

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The study neither comprised the use of human material and data nor did the research involve animals. Thus, an approval by the ethics committee was not necessary for the study.

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Guggenbichler, F., Büttner, C., Rudolph, W. et al. Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli . Appl Microbiol Biotechnol 100, 10479–10493 (2016). https://doi.org/10.1007/s00253-016-7667-5

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  • DOI: https://doi.org/10.1007/s00253-016-7667-5

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