A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry.
Protein Eng Des Sel
; 21(4): 247-55, 2008 Apr.
Article
em En
| MEDLINE
| ID: mdl-18239074
ABSTRACT
Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 10(9) variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host ( approximately 10(6) variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Staphylococcus
/
Proteínas Recombinantes de Fusão
/
Fator de Necrose Tumoral alfa
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Biblioteca de Peptídeos
/
Citometria de Fluxo
Limite:
Humans
Idioma:
En
Revista:
Protein Eng Des Sel
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2008
Tipo de documento:
Article