Self-assembled amyloid-like oligomeric-cohesin Scaffoldin for augmented protein display on the saccharomyces cerevisiae cell surface.
Appl Environ Microbiol
; 78(9): 3249-55, 2012 May.
Article
en En
| MEDLINE
| ID: mdl-22344635
ABSTRACT
In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of Saccharomyces cerevisiae is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p(1-80)). The resulting Ure2p(1-80)-HD-cohesin fusion protein was successfully expressed in Escherichia coli to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of S. cerevisiae engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the Clostridium cellulolyticum CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFP-cohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Saccharomyces cerevisiae
/
Priones
/
Proteínas Cromosómicas no Histona
/
Proteínas de Ciclo Celular
/
Proteínas de Saccharomyces cerevisiae
/
Multimerización de Proteína
/
Nanofibras
/
Glutatión Peroxidasa
/
Proteínas de la Membrana
Idioma:
En
Revista:
Appl Environ Microbiol
Año:
2012
Tipo del documento:
Article
País de afiliación:
Estados Unidos