Libraries of random peptides displayed on the surface of biological systems are an essential tool that enables systematic study of target molecule interactions. Peptide modulators of therapeutically interesting target proteins are gaining an increasingly important role in drug discovery and development. First part comprises of introduction, development and optimization of biological display libraries with the use of a model target molecule, streptavidin. The second part describes the use of such libraries and optimized procedures in a search of novel peptide ligands of selected targets. Bacterial and phage display libraries were compared for their efficiency in the search of model target protein (streptavidin) binding motif. Under similar conditions, phage display library, although technically more demanding, proved to be convincingly better. Efficiency and convenience of enzyme linked immunosorbent assay and surface plasmon resonance were compared for determining the binding properties of peptide-displaying phage clones using streptavidin as a model protein target. Both methods can be successfully used for affinity ranking. The choice therefore depends on the nature and the amount of target protein and the equipment available. Versatile sensor chips enable immobilization of almost any target molecule, regardless of their physiochemical properties and when there is only a small amount of target molecule available, surface plasmon resonance would be a method of choice. An improved non-specific elution strategy was introduced to the phage display technology. This procedure employs ultrasound to break the interaction of high affinity clones with the target and also to detach the target molecule from the immobilization surface. This new approach is able to surmount the incapability of other commonly used non-specific elution techniques and represents an important improvement in a search for novel specific ligands. Phage display technology was used to develop new ligands and potential inhibitors of lipid digestion and absorption enzymes (pancreatic lipase, pancreatic phospholipase A2) in gastrointestinal tract. A random, cyclic heptapetide and a linear dodecapeptide phage display library were used. Several independent selections were performed, differing in washing and elution steps. Inhibition of pancreatic lipase was achieved by peptide D23 (apparent inhibition constant of 16 μM). Peptide ligands of pancreatic phospholipase A2 and structurally related ammodytoxin C, selected by different selection protocols, clearly indicate affinity towards both enzymes, regardless which of the two proteins was used as a target in the selection procedure. Despite clear affinity, none of the peptides had inhibitory activity in vitro.Peptides selected and discovered by screening biological libraries target only a few sites on a given protein, often associated with biological activity. These peptides are modulators of protein function and are a starting point in drug development, their main advantage being the ability of highly specific interaction with protein macromolecules.
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