Institut für Biologie

Permanent URI for this collectionhttps://hohpublica.uni-hohenheim.de/handle/123456789/81

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Analysis of the structure of tomato mosaic virus movement protein based on virus host interactions
(2011) Tanwir, Fariha; Pfitzner, Artur J. P.
Viruses are obligatory plant pathogens causing sever diseases, and ultimately great losses in crop yield. Plant viruses, once entered in the cell, make use of host machinery for its own replication and moves from one cell to the other. Natural resistance against virus attack is achieved by the presence of resistance genes (R genes). R genes recognize viral avirulence (Avr) factors in elicitor-receptor manner to initiate resistance cascade. In tomato, the resistance genes Tm-I, Tm-2 and Tm-22 are used to protect the plants against infection by tomato mosaic virus.Tm-2 and Tm-22 require recognition of the viral 30kDa movement protein (MP) for triggering resistance response. Sequence analysis of Tm-2 and Tm-22 resistance breaking viruses have shown an amino-acid exchange at position 133 (E>K) is found in all Tm-2 resistance breaking virus strains, whereas, amino-acid exchange at position 130 (K>E) is associated with Tm-22 resistance breaking phenotype (Calder and Palukaitis, 1992). This suggests a physical interaction between resistance genes and 30kDa MP. In the present study, a unique Split GFP approach is used to analyse the structure and localization of different domains of 30kDa MP in S. cerviceae and N. benthamiana. Different deletion mutants were fused between two non-overlapping halves of GFP and expressed. Results showed that both N and C terminus as well as the middle part of 30kDa MP (aa 80-150) is present in the cytoplasm with two integral membrane loops. These findings are in contrast with previous in-vitro results, which suggest that middle part of 30kDa MP is present in ER lumen, whereas N and C terminus in cytoplasm (Brill et al., 2000). Fluorescence microscopy revealed that GFP fused 30kDa MP deletion mutants were localized on the cytoplasmic side of plasmamembrane and near plasmodesmata. Membrane association of fusion protein confirmed the proper folding and functionality of deletion mutants. Therefore, the structural model of ToMV 30kDa MP has to be revised. Secondly, to identify the host factors involved in resistance mechanism, initiated by Tm-2 and Tm-22 resistance genes, tomato mosaic virus based vectors were constructed. Two different types of in-vivo transcription vectors were constructed, one containing both right and left border of the T-DNA (pBinSLN) and one without the right border (pBinSLN-RB). Self replication of these vectors were analysed in N. benthamiana, N. tabacum and S. lycopersicum. It was found that the deletion of RB does not affect virus replication, when agro-infiltrated in N. benthamiana. pBinSLN-RB was used further for the isolation of a stable and vigorous Tm-2 and Tm-22 resistance breaking ToMV strain through a novel selection scheme. ToMV2-22 contains two amino-acid exchanges at position 54(N>D) and 133(E>K). ToMV2-22 is the first mutant strain of ToMV, which can escape both Tm-2 and Tm-22 resistance simultaneously.
Recombinant production and characterization of metalloproteins from bacterial pathogens and the innate immune response
(2024) Göbel, Katharina; Fritz, Günter
The challenges and potential solutions of drug development are highlighted by discussing the identification, production and characterization of potential new drug targets in this study. The successful development of new and specific pharmaceuticals requires that the target for the respective new drug is available as a pure and homogenous molecule in its native state. Typically, the target molecule is a protein. E.g. antibacterial drugs target proteins from a bacterial pathogen or in human diseases pharmaceuticals predominantly target proteins of signaling pathways or receptors. These proteins are usually not available directly from the organism itself and have to be produced in an expression host and purified to homogeneity. Despite the advances in the field of recombinant protein expression and purification many proteins are very difficult to produce and thus represent the major bottleneck in the development of new pharmaceuticals. In particular demanding is the expression of metalloproteins, which make up to 30% of all proteins coded in the human genome and represent a major challenge in recombinant protein production. Metalloproteins are a diverse class of proteins that is crucial for various biological processes. They play an important role in the regulation, catalysis, and maintenance of biomolecular structure. Alone, 10% of all human proteins contain zinc ions and 2% contain iron, and both metal ions are often inserted by specific but so far unknown chaperones impeding the recombinant production of correctly folded and active proteins. The challenges in studying these metalloproteins arise from their complex structures and the difficulty of their expression and isolation. To overcome these problems new approaches and solutions are highlighted and exemplified by the production and characterization of potential new drug targets in this study. The focus lies particularly on metalloproteins that play a role in infectious diseases. Global health challenges include the persistent threat of infectious diseases despite advances in healthcare, hygiene and therapeutics. The COVID-19 pandemic and rising antibiotic resistance are prime examples of the ongoing risks. This research focuses on three different proteins: (1) the maturation factor NqrM from the bacterial pathogen Vibrio cholerae, (2) the human regulator of the interferon response ubiquitin-specific protease 18 (USP18) and its interaction partners, as well as (3) the viral Papain-like protease (PLpro) from the pathogenic virus SARS-CoV-2. All three proteins belong to the class of metalloproteins and bind either iron as in the case of NqrM or zinc as for USP18 and PLpro. New methods and strategies were developed to produce, isolate and investigate these metalloproteins and since all three proteins represent potential drug targets the results presented here provide the basis for future drug development. The production of proteins requires the selection of appropriate expression host systems such as bacteria, yeast, mammalian cells, etc., depending on the desired application. The study emphasizes the versatility of expression host E. coli due to its well-studied genetics, rapid growth kinetics and ease of handling. However, challenges such as the lack of post-translational modifications can lead to the production of non-functional proteins. Optimization of expression strategies is crucial, and the study describes various factors affecting protein production, including protein engineering, growth conditions, media composition and induction parameters expanding and enhancing the well-established E.coli expression system also for very challenging target proteins. The successful isolation of the proteins formed the fundamental basis for a detailed functional and structural characterization of the proteins. The research presented here takes a forward approach and encompasses the new strategies in cloning, recombinant expression and purification of proteins from bacteria, viruses and humans, emphasizing the advantages and disadvantages of homo- and heterologous recombinant expression. The results obtained highlight also the need for extensive experimental testing to establish optimal conditions, particularly for challenging proteins such as the metalloproteins studied here.

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