Application of immobilized bovine enterokinase in repetitive fusion protein cleavage for the production of MUC1
| Autor: | T. Kubitzki, Stephan Lütz, Thomas Noll, Marco Oldiges, D. Minör, Ursula Mackfeld |
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| Přispěvatelé: | Institut für Biotechnologie 2, Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Technische Fakultät, Universität Bielefeld, GDC/PSB/BIR, Novartis |
| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
0106 biological sciences
Enteropeptidase Recombinant Fusion Proteins CHO Cells Cleavage (embryo) 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences Cricetulus Cricetinae 010608 biotechnology Protein purification Escherichia coli Animals Peptide bond Magnesium Peptide sequence 030304 developmental biology Serine protease 0303 health sciences biology Chemistry Mucin-1 Life Sciences General Medicine Enzymes Immobilized Fusion protein Immunoglobulin Fc Fragments Biochemistry Immunoglobulin G biology.protein Molecular Medicine Cattle Target protein |
| Zdroj: | Biotechnology Journal Biotechnology Journal, Wiley-VCH Verlag, 2009, 4 (11), pp.1610. ⟨10.1002/biot.200900049⟩ |
| ISSN: | 1860-6768 1860-7314 |
| DOI: | 10.1002/biot.200900049⟩ |
| Popis: | International audience; Bovine enterokinase is a serine protease catalyzing the hydrolysis of peptide bonds, which plays a key role in mammalian metabolism. Because of its high specificity towards the amino acid sequence (Asp)4-Lys, enterokinase is a potential tool for the cleavage of fusion proteins, which gain more importance in biopharmaceutical production. A candidate for adaptive cancer immunotherapy is mucin 1, which is produced recombinantly as fusion protein in CHO cells. Here, we present the first repetitive application of immobilized enterokinase for the cleavage of the mucin fusion protein. The immobilization enables a facile biocatalytic process due to simplified separation of the biocatalyst and the target protein. Immobilized enterokinase was applied in a maximum of 18 repetitive reactions. The enzyme utilization (total turnover number) was increased significantly 419-fold compared to unbound enzyme by both immobilization and optimization of process conditions. Slight enzyme inactivation throughout the reaction cycles could be observed, but was compensated by adjusting the process time accordingly. Thus, complete fusion protein cleavage was achieved. Furthermore, we obtained isolated MUC1 with a purity of more than 90% by applying a simple and efficient purification process. The presented results demonstrate EK to be an attractive tool for fusion protein cleavage. |
| Databáze: | OpenAIRE |
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