Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates
✍ Scribed by H. L. Ball; P. Mascagni
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 182 KB
- Volume
- 3
- Category
- Article
- ISSN
- 1075-2617
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✦ Synopsis
Chaperonin 10 protein from Rattus norvegicus (Rat cpn10) 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 a -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 cpn10 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 a -terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of sidechain 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.