Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis.
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.
Swennen
Dominique
D
Laboratoire de Génétique Moléculaire et Cellulaire, INA-PG, INRA UR216, CNRS URA1925, BP01 F-78850 Thiverval Grignon, France1.
Paul
Marie-Françoise
MF
Aventis Pharma France, 13, quai Jules Guesde - BP14, 94403 Vitry sur Seine, France2.
Vernis
Laurence
L
Laboratoire de Génétique Moléculaire et Cellulaire, INA-PG, INRA UR216, CNRS URA1925, BP01 F-78850 Thiverval Grignon, France1.
Beckerich
Jean-Marie
JM
Laboratoire de Génétique Moléculaire et Cellulaire, INA-PG, INRA UR216, CNRS URA1925, BP01 F-78850 Thiverval Grignon, France1.
Fournier
Alain
A
Aventis Pharma France, 13, quai Jules Guesde - BP14, 94403 Vitry sur Seine, France2.
Gaillardin
Claude
C
Laboratoire de Génétique Moléculaire et Cellulaire, INA-PG, INRA UR216, CNRS URA1925, BP01 F-78850 Thiverval Grignon, France1.
D005656:Fungal Proteins; D005822:Genetic Vectors; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism; D043484:Proprotein Convertases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics; D013381:Subtilisins / Q000378:metabolism; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism; D018631:ras Proteins / Q000276:immunology*
D005656:Fungal Proteins; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005087:Glucan 1,4-alpha-Glucosidase; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D018631:ras Proteins