Capillary electrophoresis-based biomolecular recognition studies / Estudos de reconhecimento biomolecular por eletroforese capilar

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

This Thesis concerns on the development of capillary electrophoresis-based bioanalytical methods for two distinct applications: DNA-protein binding analysis and monitoring of enzyme-catalyzed GTP hydrolysis. In the first chapter, the feasibility of on-chip electrophoretic mobility-shift assays (EMSA) is investigated. Purified transcription factors c-Jun(AP1) and p-50(NFkB) were used for binding studies to dsDNA probes containing the consensus sequences from AP1, NFkB and AP2 regulatory sequences. DNA probes were modified at the 5?-end with the Cy5 and unlabeled oligos were used for competition experiments. On-chip-EMSA could be carried out within ca. 2 h with low sample consumption and no need to handle radioactive material. Both, the dsDNA probes and the shifted oligos from binding reactions were analyzed on the ?2100 Bioanalyzer? using either, the standard procedure for DNA analysis or a modified protocol, in which no intercalating dye was used. Instead, 4.9 nM Cy5-dCTP was added to the gel matrix allowing the detection of only Cy5-labeled DNA. Despite the need of specific adjustments for each protein, we have shown the potential for replacing slab gel-based EMSA for on-chip methods. A competition experiment to show sequence specific binding of the transcription factor p-50 to the consensus sequence NFkB is presented as a proof of principle. In chapter II, a capillary electrophoresis-based method for in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4 /Bradeion ? (GST-rDGTPase). The application example demonstrates that the capillary electrophoresis technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: vmax = 1.7 μM min-1 ± 0.1 and Km = 1.0 mM ± 0.3; kcat = 9 x 10-3 s-1. In addition the effect of co-factors such as Mg2+ and Mn2+ in the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze di- and triphosphated forms of other nucleotides.

ASSUNTO(S)

interações dna-proteína transcription factors septinas bioanalyzer bioanalyzer gtp septines gtp emsa modility-shift gtpases emsa gdp dna-protein interaction fatores de transcrição gdp gtp-ase

Documentos Relacionados