A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris.
Yeast
; 28(3): 237-52, 2011 Mar.
Article
en En
| MEDLINE
| ID: mdl-21360735
ABSTRACT
To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Pichia
/
Ingeniería de Proteínas
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Retículo Endoplásmico
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Glucosiltransferasas
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Aparato de Golgi
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Manosidasas
Idioma:
En
Revista:
Yeast
Asunto de la revista:
MICROBIOLOGIA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Estados Unidos