Proefschrift Rijksuniversiteit Groningen. / Datum laatste controle: 12-11-1996. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17598号 / 工博第3757号 / 新制||工||1573(附属図書館) / 30364 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 森 泰生, 教授 跡見 晴幸 / 学位規則第4条第1項該当

Green fluorescent protein (GFP) fusion tags are commonly used to study protein expression and cellular localization in vivo. But, GFP must undergo an autogenic post-translational modification, known as chromophore maturation, to become fluorescent, a process that can have a half-time longer than 30 minutes inside research model organisms. The timescale of chromophore maturation in GFP is thus slower than many key biological processes, limiting its usefulness in measuring those processes. In this thesis, we discuss the creation and engineering of a sensor for transiently expressed proteins (STEP) based on a fully matured but dim GFP. Upon specific binding of STEPtag, a small (15.5 kDa) protein to the sensor, full fluorescence is restored. Thus, by genetically fusing STEPtag to a protein of interest, it can be detected as soon as folding is complete, without any maturation delay. Through a combination of rational design and targeted directed evolution, we describe the improvement of the original sensor, gSTEP0, into an optimized version, gSTEP1. The sensor has been validated in vitro and in E. coli cells, and we have found that for gSTEP1, the fluorescence signal increases more than three-fold upon binding, with a Kd of 120 ± 30 nM and a kon of 1.7 x 105 M-1s-1, allowing detection of the protein of interest on the second timescale. We have also created a yellow version of the biosensor, and provide preliminary attempts at developing orthogonal binding pairs, as well as red- and cyan-coloured STEPs, which could eventually be used in multiplex experiments. Our biosensor opens the door to the study of short-timescale processes in research model organisms, such as Drosophila and zebrafish embryogenesis, as well as in host-pathogen interactions, which we are currently investigating.

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Efficient delivery of vaccines and therapeutic agents in vivo is a critical aspect for ensuring a desired immunological or biological response is achieved. This work focuses on developing effective delivery systems for vaccines and therapeutics to achieve biological potency while maintaining patient friendly administration. Traditional vaccines are administered via parenteral injection, which requires skilled personnel for administration and does not elicit strong mucosal immune responses. An alternative approach is to develop an oral vaccine; however, this requires antigens to be protected during transit through the gastrointestinal tract and be transported across specialized intestinal sampling cells called M cells. These M cells are extremely rare, making them an important target for oral vaccines. The protein invasin, from Yersinia psuedotuberculosis, naturally binds [mathematical symbols] integrin, a receptor found exclusively on M cells within the gastrointestinal tract. Therefore, we generated combinatorial libraries of invasin, followed by a directed evolution and high-throughput screening strategy to identify invasin variants with increased affinity towards [mathematical symbols] integrin. This process led to the creation of an invasin variant exhibiting a nine-fold decrease in EC₅₀, which could be used for targeted oral vaccine systems. In order to test for increased vaccine efficacy due to the engineered invasin ligand, we developed a polymeric microparticle delivery system. These microparticles were formulated to encapsulate the model antigen ovalbumin and be decorated with the invasin targeting ligands. To measure physiological trafficking and intestinal retention, novel fluorescent nanocrystals were loaded into particles conjugated to invasin. These nanocrystals served as a contrast agent for in vivo imaging in mice. While these particles were unsuccessful in generating an antibody response toward ovalbumin when administered to mice, a response directed to the targeting ligand itself was observed. These findings provide insights for further optimizing a delivery system for oral vaccination. In addition to developing an oral vaccine delivery system, we created a high concentration therapeutic protein formulation, suitable for low-volume subcutaneous administration. By adding crowding agents, we were able to generate reversible protein nanoclusters with low viscosity. These nanoclusters were found to revert to monomer upon dilution and pharmacokinetic profiles similar to solutions. / text

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The formation of large, complex structures from small building blocks through self-assembly is widely seen in proteins and provides a tool for the creation of functional nanoscale devices. However, the factors controlling protein self-assembly are complex and often poorly understood. Peroxiredoxins are a large family of proteins, many of which are able to form a variety of large structures from a small, basic unit. This assembly has been shown to be strongly influenced by the redox state of the enzyme, which functions as a switch, controlling self-assembly. This thesis uses a protein from this family, human peroxiredoxin 3 (hPrx3) as a model system to investigate whether the self-assembly properties of hPrx3 can be influenced by rational protein engineering. Three forms of hPrx3 were purified and examined. These were the wild type and two variants: a mutant (S78A) and a His-tagged form. Size exclusion chromatography showed that each form showed a different ratio of dimers and larger species. Both variants showed preference for larger species, especially in the His-tagged form. This was shown to be partially dependent on metal binding in the His-tagged form. Larger species formed from multiple rings were also identified. SAXS measurement indicated that in the wild type enzyme, higher order species were dodecameric rings. For the His-tagged variant, SAXS measurement showed that the species observed had a different structure than that of the wild type. Electron microscopy showed that higher order structures seen in both wild type hPrx3 and His-tagged hPrx3 were ring shaped, with dimensions consistent with dodecamers. A competitive assay showed that the wild type, with kcat/km values near 2 x 10⁷, consistent with published results. Both variant forms showed evidence of slightly higher activity than the wild type, indicating a link between activity and assembly. A peroxiredoxin from the thermophilic bacteria Thermus aquaticus, TaqPrx was also examined, in an attempt to investigate a peroxiredoxin capable of self-assembly at high temperatures, which would be very useful for a nanoscale device. TaqPrx was cloned, purified and examined, however, no evidence of self-assembly was observed. Protein modelling and dynamic light scattering measurement indicated that the protein purified was monomeric and had a structure. Sparse matrix crystal screening identified conditions that allowed crystal formation, although strongly diffracting crystals were not produced. A novel assay for peroxiredoxin activity was developed, and suggested that TaqPrx shows peroxiredoxin activity. This thesis shows that peroxiredoxins are a useful model system for the investigation of how protein self-assembly is controlled, and how it can be influenced by protein engineering.