Engineering a switchable toxin: the potential use of PDZ domains in the expression, targeting and activation of modified saporin variants.
Protein Eng Des Sel
; 23(2): 61-8, 2010 Feb.
Article
en En
| MEDLINE
| ID: mdl-19933699
ABSTRACT
A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
Inhibidores de la Síntesis de la Proteína
/
Ingeniería de Proteínas
/
Proteínas Inactivadoras de Ribosomas Tipo 1
/
Dominios PDZ
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Eng Des Sel
Asunto de la revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Año:
2010
Tipo del documento:
Article
País de afiliación:
Italia