Expression of single-chain Fv-Fc fusions in Pichia pastoris.
J Immunol Methods
; 251(1-2): 123-35, 2001 May 01.
Article
em En
| MEDLINE
| ID: mdl-11292488
ABSTRACT
Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life. We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The increased size of the dimer (approximately 106 kDa vs. approximately 25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t(1/2) of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv.
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Bases de dados:
MEDLINE
Assunto principal:
Pichia
/
Fragmentos Fc das Imunoglobulinas
/
Fragmentos de Imunoglobulinas
Limite:
Animals
/
Humans
/
Male
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2001
Tipo de documento:
Article
País de afiliação:
Estados Unidos