Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates.
J Pept Sci
; 3(4): 252-60, 1997.
Article
en En
| MEDLINE
| ID: mdl-9262643
ABSTRACT
Chaperonin 10 protein from Rattus norvegicus (Rat cpn 10) has been reported to bind chaperonin 60 from Escherichia coli (GroEL) in an ATP-dependent manner. Chemically synthesized Rat cpn10 was immobilized in a defined orientation to agarose-bound monomeric avidin using a reversible biotinylated affinity label (1), attached to the N alpha-terminal residue. The resulting affinity chromatographic matrix was then used to isolate binding proteins from a crude cell lysate. Following affinity separation the bound ligand and ligate was released by treatment with organic base. Rat cpn 10 was prepared using a highly effective synthetic protocol involving HBTU/HOBt activation and capping with N-(2-chlorobenzyloxycarbonyloxy) succinimide to terminate unreacted amino groups. The biotinylated Fmoc-based molecule (1) was introduced specifically onto the N alpha-terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of side-chain protecting groups using a low-TFMSA/high-HF procedure. Crude biotinylated Rat cpn10 (Rat cpn10 + 1) was immobilized on monomeric avidin with a binding efficiency of approximately 75% and unlabelled truncated/capped impurities eluted off the column with buffer. The biotinylated Rat cpn10-avidin affinity matrix was then used to isolate GroEL from a crude cell lysate. The identity of the purified protein was confirmed by SDS-PAGE and binding to a specific anti-GroEL monoclonal antibody (MoAb). These results extend the applicability of the biotinylated label (1), providing a reversible non-covalent anchor for immobilization of peptide and protein ligands, thus simplifying isolation of ligates and enabling recovery of synthetic material under mild conditions.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Péptidos
/
Biotina
/
Chaperonina 60
/
Chaperonina 10
Tipo de estudio:
Guideline
Límite:
Animals
Idioma:
En
Revista:
J Pept Sci
Asunto de la revista:
BIOQUIMICA
Año:
1997
Tipo del documento:
Article
País de afiliación:
Italia
Pais de publicación:
ENGLAND
/
ESCOCIA
/
GB
/
GREAT BRITAIN
/
INGLATERRA
/
REINO UNIDO
/
SCOTLAND
/
UK
/
UNITED KINGDOM