Despite remarkable progress in understanding biology and disease at the level of nucleic acids, insights into the relevant biochemical processes frequently remain preliminary, since much regulation and activity occurs at the protein level through control of gene expression and variations of protein conformation. In particular, the effect of such variations on protein interactions is critical for a better description of biology and disease. Protein microarray technology provides a means to such ends and is a growing field of proteomics, with a high potential for analytical and functional applications in biology and medicine. On the basis of sequence information from individuals, it is possible to characterize disease-specific protein isoforms that result from mutations, polymorphisms, and splice variants with personalized protein microarrays. During my thesis, I developed such a technique. As a first step, solid-phase PCR is applied to copy a particular tissue’s RNA/cDNA onto the microarray surface, using for each gene a specific primer pair that is attached to the chip surface. The generated DNA templates are firmly attached to and specifically oriented on the array surface. The solid-phase PCR successfully amplified DNA of up to 3 kb, also allowing multiplex amplification of DNA. The arrayed DNA copies then act as templates for an in situ cell-free expression, yielding a protein microarray that presents the protein content of a particular tissue of an individual person. Expression control was conducted by a multiple spotting technique (MIST). C-terminus detection showed that translation was complete, yielding full-length proteins. During the process of setting up the technique of producing individualized protein microarrays, the MIST technology was optimized concomitantly. The various steps involved were analyzed to determine optimal conditions for template preparation, protein expression and interaction detection. Protein microarrays of 3500 human proteins were produced with these procedures and their performance was tested in model studies of protein–protein interactions.
|Supervisor:||Wiemann, Prof. Dr. Stefan|
|Date of thesis defense:||11 March 2014|
|Date Deposited:||25 Mar 2014 09:13|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||500 Natural sciences and mathematics|
|Controlled Keywords:||protein microarrays, interaction, solid-phase|