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Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation. / Master of Science

The baculovirus expression vector system (BEVS) is an effective way to produce recombinant proteins for biopharmaceuticals. However baculovirus stocks are stored in subzero temperatures to maintain virus stability, and fetal bovine serum is commonly used in the storage solution. In an effort to lower transportation and storage costs, a storage formulation that can effectively store the baculovirus in above frozen temperatures without the use of FBS would be beneficial. In this study, DMSO, ethylene glycol, glycerol, sucrose, sorbitol, sucrose-phosphate, and sucrose-phosphate-glutamate were added to baculovirus stock at various concentrations to determine the most effective stabilizer for virus storage at 4°C. Of the seven additives studied, 1 M sorbitol most effectively preserved baculovirus stock over a period of 47 weeks stored in 4°C. Formulations that include sucrose, L-arginine, and Pluronic F68 were created to determine their effectiveness on virus stability in a freeze-dried state stored at room temperature. In a lyophilized state, 0.5 M sucrose maintained baculovirus stock stability after 5 weeks of storage. Lyophilized stocks not containing sucrose were no longer infective after 5 weeks. / Master of Science

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • The UPLC-MS/MS analyses and quantification of novel 5α-reduced steroids using response factors. • The kinetic characterisation of human steroid 5α-reductase type 1 (SRD5A1), expressed in HEK-293 cells, towards 11OHA4 and 11OHT and their keto derivatives by progress curve analysis. • The subcloning, transformation and functional expression of SRD5A1 in the yeast expression system, P. pastoris. • The conversion of 11OHA4 and 11OHT and their keto derivatives by SRD5A1 expressed in P. pastoris. • The endogenous enzymatic activity in P. pastoris towards the 5α-reduced metabolites in the 11OHA4- and alternate 5α-dione pathways. • The potential application of P. pastoris as a biocatalyst in the production of 5α- reduced C19 steroids. / AFRIKAANSE OPSOMMING: Hierdie ondersoek beskryf: • Die UPLC-MS/MS analise en kwantifisering van nuut-ondekte 5α-gereduseerde steroïede met behulp van responsfaktore. • Die kinetiese karakterisering van menslike steroïed 5α-reduktase tipe 1 (SRD5A1), uitgedruk in HEK-293 selle, vir 11OHA4 en 11OHT en hul ketoderivate deur middel van progressiekurwe-analise. • Die subklonering, transformasie en funksionele uitdrukking van SRD5A1 in die gis P. pastoris. • Die omsetting van 11OHA4 en 11OHT en hul ketoderivate deur SRD5A1 uitgedruk in P. pastoris. • Die omsetting van 5α-gereduseerde steroïede in die 11OHA4 en alternatiewe 5α-dioon paaie deur endogene ensieme in P. pastoris • ‘n Ondersoek na die toepassing van die gisuitdrukkingstelsel as ‘n moontlike OR potensiële biokatalis vir die produksie van 5α-gereduseerde C19 steroïede.

The concept of introducing engineering principles of abstraction and standardization into synthetic biology has received increasing attention in the past several years and continues to be in the forefront of synthetic biology. One direction being pursued by synthetic biologists is creation of modular biological parts (BioBrickTM) that can be readily synthesized and mixed together in different combinations. However, most standard BioBrickTM parts in the Registry were designed for E. coli, although synthesis of specific BioBrickTM parts for other bacteria, such as for yeast and cyanobacteria, have begun. Besides, at the present time, there are only three chassis, which include E. coli, Bacillus subtilis, and a cell-free chassis, available in the Registry. Thus, the choices of BioBrickTM chassis are very limited. In addition, most BioBrickTM parts in the Registry have not been characterized. In the present study, the BioBrickTM concept was extended to the photosynthetic bacteria, Rhodobacter sphaeroides. In order to do that, a BioBrickTM compatible gene expression system was designed to convert R. sphaeroides to potential solar powered bio-factories or bio-refineries. This gene expression system was composed of BioBrickTM promoters, Ribosome Binding Sites (RBSs), and terminators in a BioBrickTM compatible cloning vector and its function has been validated through the expression of fluorescent proteins. In addition, a bioluminescence-based BioBrickTM characterization method was developed in this study. This method was based on a cloning vector that includes two adjacent operons, with each expressing a different luciferase reporter gene. The measured optical signals from the two expressed bioluminescent reporters were then used to predict the performance of promoters, RBSs, and terminators. Based on this bioluminescence-based BioBrickTM characterization method, two BioBrickTM characterizations kits, one for E. coli and one for R. sphaeroides, were developed. BioBrickTM parts that include seven promoters, six RBSs, and six terminators were characterized using the E. coli characterization kit. R. sphaeroides BioBrickTM parts were characterized when R. sphaeroides containing the BioBrickTM measurement constructs were cultured by both anaerobic photosynthesis and by aerobic respiration respectively. The experimental results showed that the activities of these R. sphaeroides BioBrickTM parts were very similar for the cells growing under two different conditions.

Malaria is one of the biggest current global health problems, and with the increasing occurance of drug resistant Plasmodium falciparum strains, there is an urgent need for new antimalarial drugs. Given the important role of carbonic anhydrase in Plasmodium falciparum (PfCA), it is a potential novel drug target. Heterologous expression of malaria proteins is problematic due to the unusual codon usage of the Plasmodium genome, so to overcome this problem a synthetic PfCA gene was designed, optimized for expression in Pichia pastoris. This gene was also modified to avoid glycosylation, and cloned into the vector pPICZαA under the control of the methanol inducible promoter AOX1. To facilitate export of the protein into the growth medium, the gene was fused in-frame with the α-factor secretion signal from Saccharomyces cerevisiae. The construct was successfully integrated in the genome of P. pastoris GS115, and attempts were made to express the protein and purify it using immobilized metal ion affinity chromatography.In this work, no expression of the PfCA protein could be detected, so further research should focus on optimization of expression conditions, or redesign of the expression vector.

<p>Malaria is one of the biggest current global health problems, and with the increasing occurance of drug resistant <em>Plasmodium falciparum</em> strains, there is an urgent need for new antimalarial drugs. Given the important role of carbonic anhydrase in <em>Plasmodium falciparum</em> (PfCA), it is a potential novel drug target. Heterologous expression of malaria proteins is problematic due to the unusual codon usage of the <em>Plasmodium </em>genome, so to overcome this problem a synthetic PfCA gene was designed, optimized for expression in <em>Pichia pastoris</em>. This gene was also modified to avoid glycosylation, and cloned into the vector pPICZαA under the control of the methanol inducible promoter AOX1. To facilitate export of the protein into the growth medium, the gene was fused in-frame with the α-factor secretion signal from <em>Saccharomyces cerevisiae</em>. The construct was successfully integrated in the genome of <em>P. pastoris</em> GS115, and attempts were made to express the protein and purify it using immobilized metal ion affinity chromatography.In this work, no expression of the PfCA protein could be detected, so further research should focus on optimization of expression conditions, or redesign of the expression vector.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22665号 / 農博第2420号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5296(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Infections by human schistosomes, in particular Schistosoma mansoni, account for significant morbidity and mortality every year in tropical and sub-tropical areas. The eggs of the parasite induce pathological changes in the infected host; in chronic and heavy infections, these changes may lead to death. A well-designed anti-schistosomal vaccine, alone or in concert with existing control measures such as chemotherapy, may prove to be a safe, inexpensive and effective means of reducing the occurrence of severe disease and death in S. mansoni infection. Previous studies have demonstrated the importance of the syncytial layer containing the apical plasma membrane (APM) of S. mansoni in both the survival of the parasite in the mammalian host and as a potential source of immunogens which may be utilized as vaccine candidates. In this paper we present evidence for the protective capacity of several schistosomal antigen preparations, including a calcium binding protein of the APM, S. mansoni calpain (GenBnnk accession no. M74233). We have constructed and characterized expression of a recombinant baculovirus expressing the large subunit of S. mansoni calpain, Sm-p80. This recombinant Sm-p80 is recognized by IgA, IgM, IgG1, and IgG3 isotype antibodies found in S. mansoni-infected human sera and partially-purified recombinant Sm-p80 provided a 29-39% reduction in worm burden in immunized mice challenged with S. mansoni. Our data indicate that Sm-p80 may be a useful vaccine antigen for the reduction of the morbidity associated with S. mansoni infections of mammalian hosts.

Ellipticine is an alkaloid with antitumor activity, whose mechanism of action is based on intercalation into DNA, inhibition of topoisomerase II and formation of covalent adducts with DNA, after its enzymatic activation by cytochromes P450 and/or peroxidases. Ellipticine is oxidized by cytochromes P450 to form up to five metabolites (7-hydroxy-, 9-hydroxy, 12- hydroxy-, 13-hydroxyellipticine and N2 -oxide ellipticine). 9-Hydroxy- and 7- hydroxyellipticine are considered to be detoxification metabolites, whereas 12-hydroxy-, 13- hydroxyellipticine and N2 -oxide of ellipticine are considered as activation metabolites, which are responsible for formation of covalent DNA adducts. The aim of this thesis was to examine the efficiency of human recombinant cytochromes P450 expressed in eukaryotic (SupersomesTM ) and two prokaryotic expression systems (Bactosomes) in oxidation of ellipticine. Cytochromes P450 expressed in prokaryotic systems differed in the amounts of "coexpressed" NADPH:CYP reductase. The resulting ellipticine metabolites were analyzed by HPLC. The results obtained in this thesis demonstrate that human cytochromes P450 2C9/2D6/2C19 expressed in prokaryotic or eukaryotic systems oxidize ellipticine to form up to four metabolites: 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and N2 -oxide...