title: Functional inclusion bodies produced in the yeast Pichia pastoris.
abstract: BACKGROUND
  Bacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria.
  
  
  RESULTS
  To overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity.
  
  
  CONCLUSIONS
  Structurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain.
authors.forename: Fabián
authors.initials: F
authors.lastname: Rueda
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 18, 1190, Vienna, Austria.|Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.affiliation: Servei de Microscòpia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sánchez-Chardi
authors.affiliation: Servei de Microscòpia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.
authors.forename: Mònica
authors.initials: M
authors.lastname: Roldán
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Villegas
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 18, 1190, Vienna, Austria.|Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Puxbaum
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain.
authors.forename: Neus
authors.initials: N
authors.lastname: Ferrer-Miralles
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain.|Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau, C/Sant Antoni Maria Claret, 167, 08025, Barcelona, Spain.
authors.forename: Ugutz
authors.initials: U
authors.lastname: Unzueta
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain.|CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Vázquez
authors.affiliation: Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140, Caldes de Montbui, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Garcia-Fruitós
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 18, 1190, Vienna, Austria. diethard.mattanovich@boku.ac.at.|Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190, Vienna, Austria. diethard.mattanovich@boku.ac.at.
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. antoni.villaverde@uab.cat.|Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. antoni.villaverde@uab.cat.|CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain. antoni.villaverde@uab.cat.
authors.forename: Antonio
authors.identifier: http://orcid.org/0000-0002-2615-4521
authors.initials: A
authors.lastname: Villaverde
mesh_terms: D055162:Biocatalysis; D004731:Endotoxins / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000502:physiology*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D004731:Endotoxins; D011994:Recombinant Proteins
keywords: Functional materials; Inclusion bodies; Pichia pastoris; Recombinant proteins