Effects of single D-amino acid substitutions on disruption of beta-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S.

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type.

Directed immobilization of peptide ligands to accessible pore sites by conjugation with a placeholder molecule.

When small ligands are immobilized onto a porous chromatography medium, only a limited number of binding sites contributes to the interaction with the target molecule. The main part of the ligand molecules is distributed on sites that are not accessible for the target protein due to steric hindrance. To direct the ligand into a well-accessible position, the ligand was conjugated to a large molecule that acted as a placeholder during the immobilization step.

Screening for peptide affinity ligands on CIM monoliths.

Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM((R)) disks.

Mutational analysis of a blood coagulation factor VIII-binding peptide.

A peptide screened from a combinatorial peptide library with the sequence EYKSWEYC performed best as a ligand for affinity chromatography of human blood coagulation factor VIII (FVIII). With this peptide immobilized on monolithic CIM columns via epoxy groups we were able to capture FVIII from diluted plasma. Rational substitution of amino acids by spot synthesis revealed that lysine and cysteine can be exchanged for almost all other proteinogenic amino acids without loss of affinity to FVIII.

Direct synthesis of peptides on convective interaction media monolithic columns for affinity chromatography.

Solid-phase peptide synthesis was performed on glycidyle methacrylate-co-ethylene dimethacrylate monoliths using Fmoc chemistry. The native epoxy groups were amino-functionalized by reaction with ethylenediamine or ammonia ions. A peptide directed against human blood coagulation factor VIII was synthesized as a model peptide.

Quantification of plasma-derived blood coagulation factor VIII by real-time biosensor measurements.

Plasma-derived blood coagulation factor VIII was analyzed in real time using biosensor technology. Monoclonal antibodies directed against the heavy and against the light chain of factor VIII were immobilized on different carboxymethyl dextran surfaces. Different factor VIII concentrations were injected over the antibody surfaces in parallel and response levels were determined from the dissociation phase at a fixed time after sample injection.