Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.
AUTOR(ES)
Vágner, J
RESUMO
Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=38645Documentos Relacionados
- Tagged versus untagged libraries: methods for the generation and screening of combinatorial chemical libraries.
- Spectrophotometric assay for enzyme-mediated unwinding of double-stranded DNA.
- A paradigm for drug discovery employing encoded combinatorial libraries.
- Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae
- Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.