Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli
| Autor: | Wolfram W. Rudolph, Florian Guggenbichler, Florian Gunzer, Carolin Büttner, Christoph Pöhlmann, Kurt Zimmermann |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Signal peptide Recombinant Fusion Proteins Gene Expression Peptide Biology medicine.disease_cause Applied Microbiology and Biotechnology law.invention 03 medical and health sciences law Gene expression Escherichia coli medicine Cloning Molecular chemistry.chemical_classification 030102 biochemistry & molecular biology Promoter General Medicine Fusion protein Molecular biology Interleukin-10 Molecular Weight 030104 developmental biology Biochemistry chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA Phosphorylation Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology. 100:10479-10493 |
| ISSN: | 1432-0614 0175-7598 |
| DOI: | 10.1007/s00253-016-7667-5 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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