To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.   In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.   The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors. / QC 20101102

Specific antibodies are invaluable tools for proteomics research. The availability of thoroughly validated antibodies will help to improve our understanding of protein expression, localization and function; fundamental processes and features of all living organisms. The objectives of the studies in this thesis were to develop high-throughput methods to facilitate the generation and purification of monospecific antibodies, and to address problems associated with antigen selection for difficult target proteins and subsequent validation issues. In the first of the studies, it was demonstrated that antibodies specific to human proteins could be generated in a high-throughput manner using protein epitope signature tags (PrESTs) as both antigens and affinity ligands. A previously developed purification process was adapted to a high-throughput format and this, in combination with the development of a protein microarray assay, resulted in monospecific antibodies that were used for profiling protein expression in 48 human tissues. Data obtained in these analyses suggest that a complete Human Protein Atlas should be attainable within the next ten years. In order to reduce the number of animals needed for such a massive project, and improve the cost-efficiency of antibody generation, a multiplex immunization strategy was developed in a further study. Antisera from rabbits immunized with mixtures of two, three, five and up to ten different PrESTs were successfully purified and analyzed for specificity using protein arrays. Almost 80% of the animals immunized with up to three PrESTs yielded antibodies towards all the PrESTs administered, and they yielded comparable immunohistochemical staining patterns (of consecutive human tissue sections) to those of antibodies obtained from traditional single PrEST immunizations. Proteins with highly similar sequences to other proteins present a major challenge for the proteome-wide generation of antibodies. In another study, Cytokeratin-17 which displays high sequence similarity to closely related members of the intermediate filament family, was used as a model and the specificity and cross-reactivity of antibodies generated against this target were investigated using epitope mapping in combination with comparative IHC analyses. Antibodies identified by epitope mapping as binding to the most unique parts of the Cytokeratin-17 PrESTs also showed the most Cytokeratin-17-like staining pattern, thus further supporting the strategy of using sequence identity scores as the main criteria for PrEST design. An alternative antigen design strategy was investigated for use in raising antibodies towards G-proteincoupled receptors (GPCRs). The extracellular loops and N-terminus of each of three selected GPCRs were assembled to form single antigens and the resulting antibodies were analyzed by flow cytometric and confocal microscopic analyses of cell lines over-expressing the respective receptors. The results from both flow cytometric and immunofluorescence analyses showed that the antibodies were able to bind to their targets. In addition, the antibodies were used successfully for the in situ analysis of human brain and pancreatic islet cells. / QC 20100727