O6-alkylguanine-DNA alkyltransferase as a new tool for functional proteomics

Recently the sequencing project of the human genome has been completed. The interest of the post-sequencing-era is shifting nowadays towards the investigation of the biological function and localization of the encoded proteins. Proteins are essential parts of all living organisms and participate in every process in the living cell. Despite their importance, the detailed function of the majority of all proteins remains still unknown. Therefore high-throughput methods are needed for the systematic examination of proteins. The first part of this thesis describes the development of a novel functional protein microarray, taking advantage of the highly selective reaction of the human O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. By functionalization of otherwise bioinert glass surfaces with the substrate O6-benzylguanine, AGT fusion proteins can be selectively and covalently immobilized on a solid surface (Figure S1). Due to the high selectivity of the covalent immobilization the main advantage of this AGT based approach is that fusion proteins can be immobilized directly out of cell extracts without prior purification. By analyzing a set of literature known protein-protein interactions, protein-small molecule interactions and posttranslational modifications we demonstrate the feasibility of this approach (Chapter 3). Figure S1: Principle of an AGT-based protein microarray in which x can be any protein of interest Furthermore, the development of surface materials suitable for functional protein microarrays is described. The generated surfaces allow immobilization of proteins in defined orientation and density. Intrinsically bioinert polymer brushes were generated in collaboration with the Laboratory of Polymers at the EPFL. For the production of these bioinert brush-surfaces, surface-initiated atom transfer radical polymerization (SI-ATRP) was used (Chapter 4). Additionally we addressed the importance of the orientation of immobilized proteins on solid surfaces. The immobilization of fusion proteins with random orientation on aldehyde-coated glass slides was compared with immobilization in a defined orientation on O6-benzylguanine functionalyzed polymer brushes. Subsequently, retention of activity of the proteins upon immobilization on the two different surfaces was analyzed. We demonstrated that a defined orientation of immobilized proteins is advantageous over random immobilization due to the fact that proteins, immobilized using the AGT approach, are attached in a defined orientation with their active site facing the solution phase, whereas random immobilization of proteins can result in hidden active sites (Chapter 5). In the second part of this thesis the problem of insolubility of recombinant expressed proteins is addressed. We analyzed the protein expression of a set of proteins, which were prior determined to be insoluble when expressed in E. coli. The proteins were expressed as AGT fusion proteins either in E. coli or M. smegmatis, with the goal to identify a suitable host for soluble protein expression with high yield. The AGT approach was exploited to screen for expression of soluble proteins and for the determination of the expression yields. We showed that fusion to AGT does not influence the solubility of the expressed proteins and that the expression yield in E. coli does not significantly differ from the yield in M. smegmatis, where proteins were expressed with less degradation. However, we assessed E. coli. to be the more suitable host for multi-parallel protein expression due to its easy handling and fast growth (Chapter 6). The gained knowledge of this project was transferred to the investigation of proteins from Mycobacterium tuberculosis, which is presented in the last part of this thesis. A better understanding of M. tuberculosis, with the goal of unveiling and validating new therapeutic targets, is an imperative need to improve the control and treatment of tuberculosis disease. We focused in particular on the bacterial two component adaptation systems (TCS). Since these systems do not exist in mammals they present attractive candidates for new drug targets. In order to gain a better understanding of these adaptation systems we focused on one specific TCS, SenX/RegX, one of the first identified and well established TCS, which is believed to be implicated in virulence. A radioactive in vitro phosphorylation assay was developed, which allowed the identification of two new in vitro substrates for histidine kinase SenX, namely DevR and NarL. These results confirm the hypothesis that TCS might not only consist of two components, but that cross-talk between different TCS exist in order to adapt rapidly to environmental changes.


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