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Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

Laboratoire de Génétique Moléculaire et Cellulaire, INA-PG, INRA UR216, CNRS URA1925, BP01 F-78850 Thiverval Grignon, France¹
Aventis Pharma France, 13, quai Jules Guesde – BP14, 94403 Vitry sur Seine, France²

Author for correspondence: Dominique Swennen. Tel: +33 1 30815444. Fax: +33 1 30815457. e-mail: swennen{at}grignon.inra.fr

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.

Keywords: heterologous secretion, glucoamylase

Abbreviations: AEP, Yarrowia lipolytica alkaline extracellular protease; GST, glutathione S-transferase; Kex2p, Kex2 protease (Kexin, EC 3 . 4 . 21 . 61); scFv, single-chain antibody fragment(s)

^a D. Swennen and M.-F. Paul contributed equally to this work.

^b Present address: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.

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