Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis
2002; Microbiology Society; Volume: 148; Issue: 1 Linguagem: Inglês
10.1099/00221287-148-1-41
ISSN1465-2080
AutoresDominique Swennen, Marie-Françoise Paul, Laurence Vernis, Jean-Marie Beckerich, Alain Fournier, Claude Gaillardin,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoYarrowia 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-RasVal12 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.
Referência(s)