Publication
With the human genome and many other eukaryotic and procaryotic genomes sequenced, the interest in life sciences is shifting towards the examination of the function, modification and regulation of the encoded proteins. Proteins are involved in virtually all activities and processes in a cell. Despite their importance, the detailed function of a large majority of all proteins is still unknown. High-throughput methods for the systematic study of proteins are therefore urgently needed. In the first part of this thesis, a new approach for the construction of protein microarrays is presented. For this approach the highly selective reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives is exploited. By covalently linking the alkyl group of the O6-alkylguanine to the surface of a glass slide, AGT fusion proteins immobilize themselves in a highly specific manner by reacting with the immobilized substrate. The great advantage over existing methods is the fact that, due to the high selectivity of the reaction, AGT fusion proteins can directly be immobilized from cell extracts without prior purification. Figure S1. Principle of AGT-based microarrays. In the second part of this thesis, a new class of multifunctional labels is presented. Studying proteins often relies on providing the protein of interest with a unique property that allows either purification from complex mixtures or characterization in functional assays in vivo or in vitro. A method recently developed in our laboratory allows the specific labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) with synthetic probes. The development of our multifunctional labels constitutes an important extension of this approach since it leads to the labeling of AGT fusion proteins with two different probes, for example with the spectroscopic probe fluorescein and the affinity label biotin, two labels that are employed in a variety of functional assays. Figure S2. General structure of the multifunctional labels. Another common problem was addressed using these multifunctional labels. Many of the synthetic molecules used for labeling penetrate the cell membrane poorly or not at all. This is a serious problem for in vivo applications since incubation with the probes leads to little or no uptake of the substrates. To overcome this problem, a cell-penetrating peptide, an oligo-arginine peptide which is able to transport a cargo through intact cell membranes, and a normally membrane impermeable fluorophore were attached to a benzylguanine core. With this construct it was possible to transport the fluorophore into the cells and specifically label AGT fusion proteins.