caa a22 4500 605499829 CHVBK 20210128100553.0 cr unu---uuuuu 210128e20150501xx s 000 0 eng 10.1007/s00253-014-6171-z doi (NATIONALLICENCE)springer-10.1007/s00253-014-6171-z Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor [Elektronische Daten] [Erlinda Fernández, Jorge Toledo, Manuel Mansur, Oliberto Sánchez, Dayrom Gil, Yamile González-González, Emilio Lamazares, Yaiza Fernández, Francisco Parra, Omar Farnós] The yeast Pichia pastoris is one of the most robust cell factories in use for the large-scale production of biopharmaceuticals with applications in the fields of human and animal health. Recently, intracellular high-level expression of rabbit hemorrhagic disease virus (RHDV) capsid protein (VP1) as a self-assembled multipurpose antigen/carrier was established as a production process from P. pastoris. Since recovery of VP1 from the culture media implies technological and economic advantages, the secretion of VP1 variants was undertaken in this work. Conversely, extensive degradation of VP1 was detected. Variations to culture parameters and supplementation with different classes of additives were unable to diminish degradation. Strategies were then conducted during fermentations using a recombinant variant of a non-specific BPTI-Kunitz-type protease inhibitor (rShPI-1A) isolated from the sea anemone Stichodactyla helianthus. The presence of the inhibitor in the culture medium at the recombinant protein induction phase, as well as co-culture of the yeast strains expressing VP1 and rShPI-1A, led to VP1 protection from proteolysis and to production of ordered virus-like particles. A yeast strain was also engineered to co-express the rShPI-1A inhibitor and intact VP1. Expression levels up to 116mgL−1 of VP1 were reached under these approaches. The antigen was characterized and purified in a single chromatography step, its immunogenic capacity was evaluated, and a detection test for specific antibodies was developed. This work provides feasible strategies for improvements in P. pastoris heterologous protein secretion and is the first report on co-expression of the ShPI-1A with a recombinant product otherwise subjected to proteolytic degradation. Springer-Verlag Berlin Heidelberg, 2014 Protease inhibitor nationallicence rShPI-1A nationallicence Recombinant protein production nationallicence Pichia pastoris nationallicence Calicivirus nationallicence BPTI-Kunitz-type family nationallicence Fernández Erlinda Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, 10600, Havana, Cuba aut Toledo Jorge Department of Pathophysiology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut Mansur Manuel Elanco Animal Health Ireland, Finisklin Business Park, Sligo, Ireland aut Sánchez Oliberto Department of Pharmacology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut Gil Dayrom Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba aut González-González Yamile Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006, Oviedo, Spain aut Lamazares Emilio Department of Pathophysiology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut Fernández Yaiza Department of Pharmacology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut Parra Francisco Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006, Oviedo, Spain aut Farnós Omar Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, 10600, Havana, Cuba aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/9(2015-05-01), 3875-3886 0175-7598 99:9<3875 2015 99 253 https://doi.org/10.1007/s00253-014-6171-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6171-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Fernández Erlinda Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, 10600, Havana, Cuba aut NATIONALLICENCE 700 1- Toledo Jorge Department of Pathophysiology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut NATIONALLICENCE 700 1- Mansur Manuel Elanco Animal Health Ireland, Finisklin Business Park, Sligo, Ireland aut NATIONALLICENCE 700 1- Sánchez Oliberto Department of Pharmacology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut NATIONALLICENCE 700 1- Gil Dayrom Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba aut NATIONALLICENCE 700 1- González-González Yamile Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006, Oviedo, Spain aut NATIONALLICENCE 700 1- Lamazares Emilio Department of Pathophysiology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut NATIONALLICENCE 700 1- Fernández Yaiza Department of Pharmacology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile aut NATIONALLICENCE 700 1- Parra Francisco Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006, Oviedo, Spain aut NATIONALLICENCE 700 1- Farnós Omar Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, 10600, Havana, Cuba aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/9(2015-05-01), 3875-3886 0175-7598 99:9<3875 2015 99 253