The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.

The methylotrophic yeast, Pichia past oris, is one of the most respected and widely used systems today. The ability of this yeast to produce large masses of protein and metabolize methanol as a sole source of carbon and energy is attributed to the highly induceable Alcohol Oxidase I promoter (AOXI). Despite of the disperse popularity and use of this promoter over the last 15 years, little is known about the transcription controls at a molecular level. A 5'>3' deletion analysis of the AOXI promoter was perrormed to gain understanding of the promoter's regulation and provided insight to the approximate locations of the important regulatory regions. A total of 10 truncations were made unveiling two areas ofhigh activity located between positions, -257 to-235, and, -235 to -188. In addition, a 14-base pair internal deletion was made between positions, -215 to -201. This region was shown to be necessary for transcriptional activation by deletion analysis. Sufficiency studies suggested that this 14-base pair element could serve as an activator sequence in both glucose and methanol.

In effort to understand and isolate genes that are associated with protein secretion, the Lin-Cereghino laboratory at University of the Pacific created mutant strains of Pichia pastoris using the restriction enzyme mediated integration method. The mutants exhibited an unusual ability to supersecrete beta-galactosidase, due to the effects of a randomly disrupted gene by pREMI-Z. To learn more about the novel effects of the gene disruption, nine beta-galactosidase supersecreters ( bgs ) have been characterized for their phenotypes such as growth rate, cell wall integrity, and ability to produce and secrete various types of recombinant proteins. The mutants showed various population doubling times, which ranged from 1.7 to 2.4 hours. Generally, the mutants with severely diminished growth rates had much lower secretion of the reporter proteins. The mutants also showed different levels of cell wall (osmotic) defect, indicated by moderate to severe leakage of alkaline phosphatase from the vacuole. It was revealed that the cell wall defect was not necessarily associated with increased protein secretion, which suggests that the cell wall may not be a limiting barrier for the secretion of most reporter proteins. The result of the reporter study suggests that the secretion phenotypes of bgs mutants were protein specific and likely to be dependent upon the structure of the secreted protein rather than the size.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19009号 / 工博第4051号 / 新制||工||1623(附属図書館) / 31960 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 田畑 泰彦, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...

No description available.

Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in catfish. There are limitations in the current therapeutic and preventative strategies against vAh. The pathogenesis of MAS as well as the immune response of catfish to vAh infection are poorly understood. The aim of this study is to: 1) develop a dual live attenuated vaccine against MAS and enteric septicemia of catfish caused by Edwardsiella ictaluri; and 2) evaluate the vAh bacterial load and gene expression patterns in catfish tissues following vAh infection. Previously, six recombinant vAh proteins (outer membrane protein, TonB-dependent receptor, three fimbrial proteins, and an ATPase) were identified to have vaccine efficacy against MAS, and live attenuated E. ictaluri vaccine strain ESC-NDKL1 was identified as an effective vector for expressing vAh antigens. A total of 29 recombinant ESC-NDKL1 strains have been constructed with the integration of one, two, or three genes encoding vAh antigens into the ESC-NDKL1 chromosome. Vaccine efficacy of the constructed strains was evaluated in channel catfish fingerlings. Four recombinant ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL1::atpase::fimMrfG, ESC-NDKL1::fim::fimMrfG, ESC-NDKL1::tdr::fimMrfG, and ESC-NDKL1::fim::ompA) showed significant protection against MAS with relative percent of survival (RPS) values of 55.72%, 60.18%, 61.74%, and 54.81%. Four triple recombinant ESC-NDKL1 strains (ESC-NDKL1::fimMrfG::ompA::fimA, ESC-NDKL1::atpase::fimMrfG::ompA, ESC-NDKL1::fim::fimMrfG::ompA and ESC-NDKL1::atpase::tdr::fim) showed the best protection with RPS values of 77.93%, 63.18%, 67.74%, and 82.35%. To gain a better understanding of vAh pathogenesis, catfish fingerlings were intraperitoneally injected with vAh strain ML09-119. The anterior kidney, liver, and spleen were collected for determination of vAh distribution and expression of thirteen pro-inflammatory, innate, and adaptive immune-related genes using real-time PCR. Results revealed that vAh spread into catfish tissues within 2 hours and peaked at 12 hours post-infection. vAh infection initiated a strong inflammatory response in catfish tissues. Additionally, our research revealed that surviving catfish were able to develop a primary immune response and possibly generation of memory B cells against MAS. Such information will facilitate the development of vaccines and therapeutic drugs for preventing and treating MAS outbreaks in catfish aquaculture.

No description available.

Elastin-like polypeptides (ELP) are artificially designed protein biopolymers that can be produced by living organisms. These proteins have the unique ability to undergo reversible inverse phase transition, in response to changes in temperature and/or addition of chaotropic salts. Below the transition temperature (T1) , ELP is soluble in water. Increasing the temperature above Ti, ELP coacervates into an aqeous ELP-rich phase. In this thesis, this unique feature of ELP was used in for recombinant protein purification and for the formation of aqueous multiple-phase systems. For protein purification, ELP was fused with an intein and a model protein (thioredoxin), to demonstrate a simple and inexpensive approach for recombinant protein purification. The ELP tags replace the chromatographic media and the intein replaces the use of the protease in conventional methods. Using ELP tags was found to be consistent with large -scale recombinant protein production/purification by purifying an ELP tagged protein using a stirred cell equipped with a microfiltration membrane. When the temperature and/or salt concer.tration is increased for mixtures containing free ELP and ELP tagged proteins, simultaneous phase transition takes place. This served as the basis for the development of a method suitable for selectively recovering molecules from complex mixtures with high specificity, full reversibility, and virtually unlimited affinity. The second parts of this thesis focus on the ability of ELP to form aqueous twophase systems (A TPS) in vitro and most importantly, in vivo- with the formation of aqueous microcompartments in living cells. These compartments exclude the protein making machinery of the cell, acting as depots for newly expressed protein. It is also shown (in vitro) that ELP bastd droplets exclude proteases, protecting proteins from degradation. These observations are important for high-level production of recombinant proteins. Also described, is the formation of protein based aqueous multiphasic systems, with tunable morphologies. / Thesis / Doctor of Philosophy (PhD)

<p> Vaccination is arguably the most effective tool at our disposal to prevent the morbidity and mortality associated with infectious disease. However, there are currently several infectious diseases, notably HIV, malaria and tuberculosis, for which we do not posses effective vaccines. Further complicating matters, traditional methods to construct vaccines for these diseases have been unsuccessful. Advances in our understanding of adaptive immunity have demonstrated that vaccines for these diseases likely rely upon potent T cell immunity to be effective. Recombinant adenovirus (rAd) vectors have shown great promise as vaccination platforms since they are easily constructed, stable, well-tolerated and elicit robust T cell responses. The robust activity of rAd vectors based on the human serotype 5 virus (rHuAd5) in murine and simian models merits futher investigation as a prototypic T cell vaccine. To this end, we have undertaken a comprehensive evaluation of T cell immunity following rAd vaccination. Our previous observations determined that the CD8+ T cell response produced by rHuAd5 vaccines displayed a prolonged effector phase that was associated with long-lived antigen presentation. We have further investigated the mechanisms underlying the maintenance of this memory population. Our results have revealed that the memory phenotype is not due to continual recruitment of naive CD8+ T cells. Rather, the sustained effector phenotype appears to depend upon prolonged expression of the antigen-encoding transgene from the rHuAd5 vector. Interestingly, transgene expression was only required for 60 days after which point the memory population stabilized. Further investigation of the relationship between antigen structure and the CD8+ T cell response revealed that antigens which traffic through the ER produce a CD8+ T cell response that expands more rapidly and displays a more pronounced contraction phase than antigens which are produced within the cytosol. While the exact mechanism underlying this phenomenon is not known, we suspect that pathways related to ER stress may be involved. Despite the more dramatic contraction phase associated with antigens that traffic through the ER, the memory phenotype was unchanged. Interestingly, the CD4+ T cell response was not influenced by antigen structure and displays a sharp contraction phase regardless of whether the antigen traffics through the ER or is produced in the cytosol. We further investigated the relationship between CD4+ T cell help and CD8+ T cell immunity produced by rHuAd5. Based on the partially-exhausted phenotype of the CD8+ T cells produced by rHuAd5 (diminished TNF-a production and little IL-2 production), we suspected that inadequate CD4+ T cell help may have been responsible. However, removal of CD4+ T cells did not further impair the CD8+ T cell response produced by rHuAd5. Rather, a lack of CD4+ T cell help only impacted the magnitude of the primary CD8+ T cell response generated by rHuAd5; the functionality of the CD8+ T cell population, including the ability to proliferate following secondary stimulation, were not affected by the absence of CD4+ T cells. Thus, although CD8+ T cell expansion following immunization with rHuAd5 is dependent upon the availability of CD4+ T cell help, the memory functions of the CD8+ T cell population appears to be independent of CD4+ T cell help. Finally, we compared the magnitude of the CD8+ T cell response produced by rHuAd5 and recombinant vaccinia virus. Our results demonstrated that the functionality of the early T cell response produced by both vectors were identical. However, the primary transgene-specific CD8+ T cell responses produced by rHuAd5 were significantly larger than rVV because the vector specific responses were negligible in the case of rAd but very strong following rVV inoculation. This research has contributed to our understanding of T cell immunity following rAd immunization and will assist in the construction and implementation of future vaccines. </p> / Thesis / Doctor of Philosophy (PhD)