Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia Lipolytica and Kluyveromyces lactis
| Autor: | Jean-Marie Beckerich, Marie-Françoise Paul, Laurence Vernis, Claude Gaillardin, Dominique Swennen, Alain Fournier |
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| Přispěvatelé: | Microbiologie et Génétique Moléculaire (MGM), Institut National de la Recherche Agronomique (INRA)-Institut National Agronomique Paris-Grignon (INA P-G)-Centre National de la Recherche Scientifique (CNRS) |
| Předmět: |
0106 biological sciences
Saccharomyces cerevisiae Proteins Sequence analysis Recombinant Fusion Proteins Genetic Vectors Yarrowia Heterologous chemical and pharmacologic phenomena Biology 01 natural sciences Microbiology Fungal Proteins Kluyveromyces 03 medical and health sciences 010608 biotechnology Subtilisins Immunoglobulin Fragments ComputingMilieux_MISCELLANEOUS Kluyveromyces lactis 0303 health sciences Fungal protein Signal peptidase 030306 microbiology respiratory system biology.organism_classification [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology Biochemistry Arxula adeninivorans Saccharomycetales ras Proteins Proprotein Convertases Expression cassette Glucan 1 4-alpha-Glucosidase |
| Zdroj: | HAL Microbiology Microbiology, Microbiology Society, 2002, 148, pp.41-50 |
| ISSN: | 1350-0872 1465-2080 |
| Popis: | Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for the expression of heterologous proteins. An anti-Ras single-chain antibody fragment (scFv) coding sequence was fused in-frame to different pre- or prepro-regions, or downstream from a reporter secretory gene (Arxula adeninivorans glucoamylase), separated by a Kex2 protease (Kex2p)-like processing sequence. Both organisms are able to secrete soluble scFv, with yields depending on the nature of the expression cassette, up to levels ranging from 10 to 20 mg l(-1). N-terminal sequence analysis of the purified scFv showed that fusions are correctly processed to the mature scFv by a signal peptidase or a Kex2p-type endoprotease present in Y. lipolytica and K. lactis. The scFv protein also retains the capacity to bind to a glutathioneS-transferase (GST)-Harvey-Ras(Val12) fusion, indicating that the antibody is functional. These results indicate that the yeasts Y. lipolytica and K. lactis have potential for industrial production of soluble and active scFv. |
| Databáze: | OpenAIRE |
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