Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates.

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.