Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Abstract

Proteolysis of short NO-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 groupsNa_tert-butyloxycarbonyl (Boc) and Na_9-fluorenylmethyloxycarbonyl (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 105 members at -6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-.8-endorphin antibody, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Immobilization Reduces the Activity of Surface-Bound Cationic Antimicrobial Peptides with No Influence upon the Activity Spectrum

“…This might have been partly due to the different synthesis procedures and limited accessibility of the reactive functional groups. It has been reported that (at most) 15% of the total amounts of functional groups of typical TentaGel beads are located on the surface of the bead (55). Moreover, protein immobilization on TentaGel was shown to be limited to the bead surface (2).…”

Immunoreactivity of Organic Mimeotopes of the E2 Component of Pyruvate Dehydrogenase: Connecting Xenobiotics with Primary Biliary Cirrhosis

“…Each bead has 100 pmol peptide conjugate (19). Columns containing beads were first blocked with 1% BSA in PBS for 30 min.…”

Cationic Antimicrobial Peptides

“…This might be partially due to the different synthesis procedures and limited accessibility of the reactive functional groups. It has been reported that at most 15% of the total amount of functional groups of typical TentaGel beads are located on the surface of the bead [152]. Moreover, protein immobilization onto TentaGel was shown to be limited to the bead surface [153].…”

“…The comparably high MIC values of C-terminally immobilized KLAL and MK5E are likely related to the fact that the majority of these sequences (~ 75%; see ref. [152]) were tethered in the interior of the porous TentaGel S NH 2 and might not be accessible for membrane interaction [152,153]. Indeed, the small pore size of TentaGel S NH 2 [145] does not allow for bacterial entry and penetration.…”