Expression and characterization of the human endothelin-A-receptor in Pichia pastoris: influence of N-terminal epitope tags.
J Cardiovasc Pharmacol
; 36(5 Suppl 1): S55-7, 2000 Nov.
Article
em En
| MEDLINE
| ID: mdl-11078335
ABSTRACT
Knowledge of the three-dimensional structure of the endothelin-A receptor (ET(A)) will provide important information for rational drug design of antagonists and agonists. In order to produce correctly folded and active receptor protein for physical characterization by such techniques as X-ray crystallography and circular dichroism spectroscopy, we have cloned and expressed the human ET(A)-receptor in Pichia pastoris. The expression constructs all contained signal sequences to direct the expressed protein to the membrane fraction. Fidelity of folding and the subtype specificity of the expressed receptor was demonstrated by ligand binding studies using 125I-labelled endothelin-1 (ET-1). Expression of receptor with two different N-terminal epitope 'tags' had little effect on the affinity of ET-1 for the receptor. The strain of P. pastoris employed did influence the quantity of receptor expressed.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pichia
/
Proteínas Recombinantes
/
Receptores de Endotelina
Limite:
Humans
Idioma:
En
Ano de publicação:
2000
Tipo de documento:
Article