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Development of instrumentation and high-throughput screening methods for peptide ligand discovery and validation
Kryštůfek, Robin ; Konvalinka, Jan (advisor) ; Jiráček, Jiří (referee)
Peptides are used as synthetically available and easily derivatizable scaffold upon which it is possible to develop ligands targeting broad spectrum of biological targets. A time-tested approach to peptide binder identification is the preparation and screening of combinatorial libraries. Bypassing of this complicated procedure is possible by using biological systems for presentation, identification and selection of peptides based on the principle of in vitro evolution - i.e. display techniques. There are two complementary automated solutions for peptide binder identification described in this work. First is the SPENSER parallel peptide synthesizer, developed as a part of this diploma project, which can be used for peptide ligand discovery and optimization as well as validation of ligands identified using display techniques. Several libraries consisting of a total of 1 052 peptides have been prepared and then used to describe its potential applications. A sample of 154 preparations, representing 14.6 % analytical coverage of the prepared libraries, showed an average purity of 67 ± 19 % according to LC-MS. The libraries presented illustrate that SPENSER is a suitable tool for the parallel synthesis of linear and disulfide-cyclized peptides with limited variability, or libraries consisting of short...
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Design, preparation and characterisation of polymer conjugates for protein recognition using peptide ligands
Kryštůfek, Robin ; Konvalinka, Jan (advisor) ; Hořejší, Václav (referee)
Antibodies stand as a cornerstone of many laboratory assays and are used as indispensable tools in biomedicine. iBodies polymer conjugates have been proposed as a possible alternative to antibodies in several traditionally immunological methods. iBodies are based on methacrylamide copolymers decorated with affinity anchors, reporters and targeting ligands which mediate conjugate specificity. The necessity to design ligands and their linkage is a significant obstacle to the generalization of this technology. One way of overcoming this obstacle is the development of secondary antibody mimetics which will use antibodies to obtain specificity for target identification and/or visualization. Based on the literature, several cyclic peptide Fc-binding ligands were designed with linker moieties for attachment to the polymer conjugate. Free and conjugated ligands were compared based on their interaction constants with human IgG. Conjugation with polymer carrier in all cases led to an increase in affinity. Human isotype specificity is dependent on the cyclization method, with single disulfide bridge cyclization exhibiting selectivity for IgG1-4 and IgM, and N→C cyclization leading to abolition of IgM binding. The polymer conjugates have been compared in terms of sensitivity and selectivity using dot blot with...
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Design, preparation and characterisation of polymer conjugates for protein recognition using peptide ligands
Kryštůfek, Robin ; Konvalinka, Jan (advisor) ; Hořejší, Václav (referee)
Antibodies stand as a cornerstone of many laboratory assays and are used as indispensable tools in biomedicine. iBodies polymer conjugates have been proposed as a possible alternative to antibodies in several traditionally immunological methods. iBodies are based on methacrylamide copolymers decorated with affinity anchors, reporters and targeting ligands which mediate conjugate specificity. The necessity to design ligands and their linkage is a significant obstacle to the generalization of this technology. One way of overcoming this obstacle is the development of secondary antibody mimetics which will use antibodies to obtain specificity for target identification and/or visualization. Based on the literature, several cyclic peptide Fc-binding ligands were designed with linker moieties for attachment to the polymer conjugate. Free and conjugated ligands were compared based on their interaction constants with human IgG. Conjugation with polymer carrier in all cases led to an increase in affinity. Human isotype specificity is dependent on the cyclization method, with single disulfide bridge cyclization exhibiting selectivity for IgG1-4 and IgM, and N→C cyclization leading to abolition of IgM binding. The polymer conjugates have been compared in terms of sensitivity and selectivity using dot blot with...
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