purcc 2012 - University of the Pacific

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Poster Session Abstracts In Vitro Activity of Calpain Inhibitors Against Tritrichomonas foetus Alex Yee, Tiffany Riley, Asma Patel, Raquel O’Connor, Neal Patel Faculty Mentor: Kirkwood Land Calpains have been shown to have a pathogenic role in many microbial diseases. In this study, we have examined the role of calpain in the in vitro viability of the veterinary protozoal parasite Tritrichomonas foetus. Several calpain inhibitors were tested on the in vitro growth of T. foetus strain D1, the most virulent strain characterized in the laboratory. Although not as potent against T. foetus as other trichomonads, the 50% reduction in viability suggests a important role of these enzymes in viability. This finding warrants further study on role of calpains in the life cycle of this important pathogen. Effects of MAT alpha deletions on protein secretion in Pichia pastoris Kimiko Agari, Hansel Poerwanto Faculty Mentors: Joan Lin-Cereghino, Geoff Lin-Cereghino Pichia pastoris is a yeast known to efficiently express and secrete heterologous proteins. In this yeast, MAT alpha is a signal that can direct protein secretion. Its effect on secretion can be tested using reporter genes, whose protein products can readily be measured. The goal of our project was to create specific deletions in the MAT alpha secretion signal and to determine their effects on secretion. Mutant constructs were made via site-directed mutagenesis, and the reporter genes tested were horseradish peroxidase (HRP) and lipase. We hypothesized that distinct deletions in the MAT alpha secretion signal should affect protein secretion differently, and these effects were tested by comparing secretion of the HRP and lipase proteins. Visualizing the Pathways of MBP-EGFP Fusions with Fluorescence Microscopy Pachai Moua Faculty Mentors: Geoff Lin-Cereghino, Joan Lin-Cereghino The yeast Pichia pastoris is known to be efficient at expressing and producing recombinant proteins. Previous studies successfully produced the maltose binding protein (MBP), a type of "escort" protein that aids protein folding and purification. We expressed enhanced green fluorescent protein (EGFP) fused to either the N-terminus of MBP (MBP-EGFP, pJV4) or to the C-terminus MBP (EGFP-MBP, pVJ103). Surprisingly, MBP- EGFP was proteolyzed before secretion, but EGFP-MBP was secreted intact. The objective was to find out if the two fusions followed different paths in the cell by using fluorescence microscopy. This led to the development of a protocol for visualizing EGFP in Pichia pastoris cells. Our results suggest that depending on its position in the fusion, EGFP followed a different route in the cell. Messing with Perfection: Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris Maria Nattestad, Kristin Oshiro Faculty Mentors: Geoff Lin-Cereghino, Joan Lin-Cereghino Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical, and basic research purposes. In most cases, the 5' untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in the yeast. Because the effect of the AOX1 5'UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5'UTR clearly decreased expression of a beta-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5'UTR 56
Poster Session Abstracts contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5'UTR optimized for a higher level of expression may be challenging. Expression and Purification of Pyriform Spidroin 2 Protein Nadia Shaheen Faculty Mentors: Joan Lin-Cereghino, Geoff Lin-Cereghino Pichia pastoris is a yeast commonly used for expression of foreign proteins, as the yeast are easily genetically manipulated, can be grown in high concentrations, and express large amounts of heterologous proteins. In this case, Pichia pastoris was used to express the Pyriform Spidroin 2 Protein, PySp2, a spider silk attachment disk glue protein. After growth and induction of PySp2, expression of the protein was confirmed through western analysis. Expression was optimized by varying culture conditions. PySp2 was then purified from the cultures via affinity chromatography using both native and denaturing conditions. The protein was successfully expressed on small and large scales; however, purification in native conditions resulted in a low yield. The yield from denaturing conditions, on the other hand, was significantly higher. Ultimately, the properties of heterologously expressed PySp2 protein can be compared to naturally produced PySp2 protein. This will help determine whether Pichia pastoris is an ideal resource to synthesize spider silk proteins on a larger scale. The Structural Studies of Artificial Silk Fibers and the PySp2 Protein Hasan AlKazemi, Jacky Aguilar, Sophia Chou Faculty Mentor: Craig Vierra Spider silk is a biodegradable, non-toxic biopolymer that is stronger than Kelvar, Nylon, and steel. Spider silk can be used in a variety of fields, including engineering and medicine. In our research, we are attempting to spin synthetic spider silk from a glue silk protein, PySp2. PySp2, which is expressed in golden orb weavers, is spun into attachment discs and helps immobilize dragline threads. PySp2 contains internal block repeats whose sequences can be tested for their unique mechanical properties. Using genetic engineering, we inserted a segment of the PySp2 cDNA into the prokaryotic expression vector pBAD-Thio-TOPO. Restriction digestion analysis and agarose gel electrophoresis was performed to verify the presence and directionality of the PySp2 cDNA in the cloning vector. Following the confirmation of the cDNA insert in the cloning vector, we induced the expression of PySp2 in bacteria and monitored its expression using western blot analysis. Our long term goal is to purify the PySp2 protein and spin artificial silk fibers as well as use the solubilized protein for structural studies. Expression of the Latrodectus hesperus Glue Silk Protein, Pyriform Spidroin 1, in Bacteria Richard Chen, Alex Hoang-Mendoza, Jun Park, Pauline Pham, Moe Thien Faculty Mentor: Craig Vierra The biological mechanisms that spiders use to spin silk fibers remains a mystery to scientists, making it difficult from labs to biomimic this process. Spiders spin multiple silk types that have a diverse range of biological functions. The highly studied dragline silk has been shown to have tremendous properties that rival both steel and Kevlar in elasticity, toughness, and tensile strength. In this lab, the pyriform spidroin protein 1 (PySp1) from the black widow spider, Latrodectus Hesperus, was studied to help elucidate how it provides a strong, adhesive glue-like function that anchors dragline silk. The purpose of our research was to express a portion of the PySp1 protein. PCR was used to amplify a segment of the PySp1 cDNA, which was then inserted into the pBAD-Thio/TOPO bacterial expression vector. E. coli was then transformed with the ligation mixture and colonies carrying the correct vector were identifies by restriction digestion and agarose gel electrophoresis. Transformants carrying the vector were induced with arabinose to express the PySp1 cDNA insert. Western blot analysis was then used to check for the expression of the PySp1 protein. With the results obtained, further experiments will be carries out in attempt to synthesize synthetic pyriform fibers. 57
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Program & Abstracts 12 th Annual Pa
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Senior Art & Design Show - Reynolds
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