In recent years, biocatalytical steps in chemical synthesis are becoming increasingly important for economical and environmental-friendly production. In order to evaluate the use of enzymes in a process at Cambrex Karlskoga AB, an expression system was developed for a dehydrogenase. A synthetic gene was cloned into Escherichia coli DH5a cells, using the pTZ19R expression vector, as previously described in the literature. Protein expression was carried out at 25°C, 30°C and 37°C and results were measured using SDS-PAGE and activity assays. To improve expression, the gene was modified in three ways using PCR, yielding eight clones: It was inserted into the pSE420 expression vector, shortened to avoid inclusion body formation and a missing nucleotide was inserted into the sequence. A protocol for inclusion body screening was also developed. Finally, an assay for determining the kinetic constants of dehydrogenase was designed. It is concluded that further experiments must be done to obtain expression of the dehydrogenase and recommendations for additional work are given. / Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.

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This dissertation investigates harvesting of marine microalgae for bioenergy and production of two recombinant proteins for therapeutic applications in Chlamydomonas reinhardtii. The first study describes harvesting of marine microalgae by flocculation using aluminum chloride (AlCl_3), natural polymer chitosan, and synthetic cationic polymers. Harvesting and concentration process of low concentration microalgae cultures ranging from 1 to 2 g dry weight per liter was affected by algogenic organic matter (AOM), ionic strength, cell concentration, polymer charge density, and media pH. Marine microalgae flocculation was greatly affected by the presence of AOM independently of the flocculant chemistry. Presence of AOM demanded extra flocculant dosage i.e., 3-fold of AlCl3, 7-fold of highly charged synthetic cationic polymer, and 10-fold of chitosan. Flocculant dosage required for > 90 % flocculation efficiency in the presence of AOM was 160 mg/L, 50 mg/L, and 20 mg/L when using AlCl_3, chitosan, and best (more efficient) synthetic polymer respectively. The high-ionic strength of saline water did not have a significant effect on flocculation efficiency when using AlCl_3. However, to achieve efficient algal biomass removal, application of highly-charged synthetic polymers was required to overcome the presence of electrolytes. The best synthetic cationic polymer tested herein, which achieved greater than 90 % flocculation efficiency at 20 mg/L dosage, was a polymer with 99 % cationic charge density. Cell concentration also affected flocculant dosage requirement; low density cultures (10^6 cells/mL) required 6-fold greater dosages than cultures grown until early stationary phase (10^7 cells/mL). The second study addresses cultivation, extraction and purification challenges of two complex recombinant proteins, an immunotoxin molecule (MT51) and malaria vaccine antigen (Pfs25) produced in the chloroplast of C. reinhardtii. Main challenges identified were i) low transgene expression level, ii) proteolytic instability of MT51 immunotoxin, and iii) aggregation of Pfs25 antigen. Optimal expression and accumulation of Pfs25 antigen required growing C. reinhardtii cultures to late exponential phase (10^6 cells/mL) and inducing transgene expression for 24 h at a photon irradiance of 120 µmol/m^2s.

The ubiquitination process is vital to maintain the protein homeostasis in the cell. With high specificity it regulates degradation of proteins by tagging them with a small protein called ubiquitin. Four proteins are involved to perform the process and in this thesis one of these proteins is studied. This protein is called Ro52 and belongs to the TRIM protein family. It posses E3 ligase activity because of a N-terminal RING-domain and therefore it is responsible for the last step in the ubiquitination process. The structure of Ro52 is not totally solved and the function of the protein’s four domains is not fully understood. In this thesis three constructs of two domains from Ro52 (RING and B-box) is investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and auto-ubiquitination assay by Western blot. The goal was to gain deeper insight in structural and functional properties of these domains. In the end only two constructs were investigated because of time limitations. It was shown by NMR that one construct has similar structure as the wild type but lower stability, possibly due to shorter N-terminal region. Comparison of the results from CD measurements showed that the constructs were well structured but did not reveal any significant differences in secondary structure between the constructs. Functional analysis by Western blot encountered unexpected problems and no results were obtained. The current thesis provides a basis for further investigation of variant constructs jointly expressing the RING-B-box domains, and shows that even small changes may alter structure and stability in ways that might affect functional properties.

African horsesickness (AHS) is one of the most deadly diseases of horses, with a mortality rate of over 90% in horses that have not been exposed to any African horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are primarily transmitted to their mammalian hosts through certain haematophagous midge vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2 by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans, 1982). It is believed that this cleavage affects the ability of the virus to infect cells of the mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a truncated VP2. Upon further investigation, this strain was also shown to be more infective than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012). Therefore, through proteolytic cleavage of these viral particles, the ability of the adult Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel et al., 2011). Based on these findings, it is important to investigate the factors that influence the capability of arthropod-borne viruses to infect their insect vectors, mammalian hosts and their known reservoirs. In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C. imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were separated on SDS-PAGE and yielded several protein bands, one of which also had a molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel. The activity of the protein of interest was also confirmed to be a trypsin-like serine protease with the use of class-specific protease inhibitors. A recombinant trypsin-like serine protease of C. sonorensis was generated using the pColdIII bacterial expression vector. The expressed protein was partially purified with nickel ion affinity chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the expressed protein was classified as a serine protease. It was also proposed that incubation of purified AHSV4 with the recombinant protease would result in the cleavage of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after which the virus was incubated with the recombinant protease. Since not enough virus sample was obtained, the outcome of VP2 digestion was undetermined. In the last part of this study, it was postulated that C. imicola and C. sonorensis have the same trypsin-like serine protease responsible for the cleavage of VP2 based on the protease activity visualised in the whole midge homogenate. Since the genome of C. imicola is not yet sequenced, the sequence of this likely protease is still unknown. Therefore, we attempted to identify this C. imicola protease through polymerase chain reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA fragment was amplified. However, sequence alignment and the basic local alignment software tool (BLAST), revealed that DNA did not encode with any other known proteins or proteases. From the literature it seems that there is a correlation between the proteases in the vector and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al., 2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The 29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in future investigations on how proteolytic viral modifications affect infectivity between different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Leishmania are protozoan parasites that are transmitted by a sand fly vector. These parasites affect not only humans but also wild animals including domestic dogs and rodents, which form an additional challenge and public health problem to control the disease. Leishmaniasis is an important disease with worldwide distribution, including Saudi Arabia, the Middle East, and other tropical and subtropical areas around the world. Due to the expansion of irrigation and agricultural activities, more exposure to sand fly occurs, which leads to the expansion of leishmaniasis infections as newly emerging disease. Emerging drug resistance in leishmaniasis is an additional problem, contributed by enzymes involved in the detoxification of pharmacological agents and other xenobiotics. Cytochrome b5 reductase (Cb5r) has a high pharmacological significance because of its essential role in fatty acid elongation, biosynthesis of cholesterol (humans) or ergosterol (Leishmania, fungi), and cytochrome P450-mediated detoxification of xenobiotics. Leishmania Cb5r has seven different isoforms whereas human has only one. Cb5r-7 isoform in Leishmania has closest homology to the human Cb5r. The three specific aims of this thesis project are focusing on (i) cloning of the Cb5r-7 isoform from Leishmania mexicana, (ii) its purification as recombinant His-tagged protein from E.coli, and (iii) its functional characterization as potential pharmacological target against Leishmania.

Research was undertaken to discover and study trypanosome proteins that may play important roles in host-parasite or vector-parasite interactions. The methods used mass spectrometry based proteomics ideally suited for analysis of low abundance molecules. First, isobaric tags were used to monitor changes in proteins expression throughout the life cycle of Trypanosoma congolense, an economically important livestock pathogen. This was the first large scale survey of protein expression in trypanosomes. In addition to generating protein expression data for approximately 2000 different parasite proteins, 6 previously undescribed T. congolense proteins were discovered. Several of the proteins with interesting expression trends were selected for molecular characterization and monoclonal antibody derivation. Second, immunoenrichment and mass spectrometry were used to identify the cognate antigen recognized by a T. congolense-specific monoclonal antibody. The antigen, a flagellar calcium binding protein, was expressed as a recombinant protein and used to test its utility as a potential serodiagnostic antigen for diagnosis of T. congolense infections. Third, a “deep-mining” protein discovery mass spectrometric method was used to identify trypanosome proteins present in the plasma of late-stage African sleeping sickness patients. A total of 254 trypanosome proteins were unequivocally identified by tandem mass spectrometry. These findings are unprecedented since never before have such a large number of pathogen proteins been discovered in human blood using a non-biased approach (i.e. without using a targeted assay). The proteins discovered provide insights into host-parasite interactions and are strong candidates as targets for new diagnostic assays. / Graduate

Intrinsic or acquired drug resistance of tumours is a major problem for successful therapy of breast cancer patients. The efficacy of doxorubicin, one of the most important and commonly used drugs in chemotherapy, can be severely compromised by a variety of unspecific mechanisms rendering tumours drug resistant. Little is known however, about the specific events taking place in response to doxorubicin treatment, which may repair doxorubicin-induced damage, leading to drug resistance. Doxorubicin is a topoisomerase II poison, which interferes with topoisomerase II enzymes during DNA replication, resulting in DNA double-strand breaks. Topoisomerase II enzymes mediate the passage of DNA strands by introducing transient DNA breaks, and are essential for changes in DNA topology during replication. The DNA lesions induced by the combination of topoisomerase II and doxorubicin can be repaired by either non homologous end-joining or homologous recombination repair, as both pathways are specifically responsible for the repair of DNA double-strand breaks. The DNA-dependent protein kinase catalytic subunit in non homologous end-joining and Rad51 in homologous recombination repair are essential for each of these pathways. If it was possible to specifically target these proteins or other antagonistic mechanisms of doxorubicin-induced cell death, which may be activated in response to doxorubicin treatment, chemosensitivity of tumours could be restored and chemotherapy made more effective. Hence it was the purpose of this study to investigate proteome-wide changes in protein expression in response to drug treatment, as well as specifically analysing alterations in the protein levels of the DNA-dependent protein kinase catalytic subunit and Rad51. Global changes in protein regulation of breast and breast cancer cells were investigated using mass spectrometric and electrophoretic analysis techniques. These experiments however, could not reproducibly identify any genuine drug-induced changes in protein levels, as only proteins of relatively high abundance could be analysed. Immunoblotting results however, showed that Rad51 was differentially regulated in a cell line- and drug dosage-dependent manner, while levels of the DNA-dependent protein kinase catalytic subunit remained largely unchanged. Furthermore, increased levels of topoisomerase II alpha protein were also detected. In addition, immunohistochemical analysis demonstrated that both Rad51 and the DNA-dependent protein kinase catalytic subunit could be independently overexpressed in breast tumours and therefore may represent potential targets for selectively enhancing chemosensitivity of breast cancers.

White wine clarity is essential, but it can be marred by the presence of a protein haze. This protein haze is predominantly formed by grape-derived thaumatin-like proteins and chitinases, which can slowly denature and aggregate if left in bottled wines. Currently bentonite fining is used by the wine industry to prevent protein haze. Bentonite consists of fine clay particles that, when added to wine, bind and remove the haze-forming proteins. However this method is inconvenient, time-consuming, and causes significant losses of wine. It is estimated that this process costs the Australian wine industry $50 m annually in wine losses alone. Alternatives are thus being investigated. The principal objective of this thesis was to investigate the sensory effects on wine of an alternative method to bentonite fining: addition of haze protective factor 2, known as Hpf2. Hpf2 is a Saccharomyces cerevisiae mannoprotein that has been shown to reduce protein haze in wines. It is a highly mannosylated 180 kDa protein, of which approximately 75% by weight is mannose. Previous work has shown that the addition of approximately 200 mg L⁻¹ Hpf2 to wines reduces the visible haze in wine by approximately 50%. Hpf2 is naturally present in wines at concentrations of less than 10 ng L⁻¹, much lower than the concentration required for haze protection activity. However, the sensory impacts involved with the addition of such high concentrations of Hpf2 in wine have never been studied. This knowledge is essential for the future commercial prospects of this alternative approach to protein stabilisation of wine. To undertake sensory studies, over 1 g of Hpf2 would be required. Presently, the laboratory-scale process for the production of a 6-histidine tagged version of the protein, 6xHisHpf2, in a laboratory yeast strain of S. cerevisiae, produces only milligram quantities. Consequently, the first challenge of this research was to scale up the existing process to produce sufficient quantities of Hpf2. The first attempt to increase the production level was by over-expression in the bacteria Escherichia coli. Although several approaches were trialled, 6xHisHpf2 was unable to be successfully and consistently expressed in this system. The second method was by improving the original yeast expression system, and the expression level was able to be improved approximately 10-fold. This improved expression method was scaled up to produce and then purify over 1 g of protein. Several quantification methods were assessed to determine the efficiencies of each purification step, with slot blot analysis proving successful. Sensory trials were conducted to establish the effect of 6xHisHpf2 on wines, with duo-trio studies conducted assessing both aroma and palate of the wines. Invertase, another yeast haze protective factor, was also trialled. It was found that the addition of an active level of 6xHisHpf2 or invertase did not cause a significant difference in the aroma or palate of wines. In addition to this main study, the role of the glycosylation was studied. 6xHisHpf2, produced in a different yeast, Pichia pastoris, was found to be 83 kDa, with only 50% mannose. This protein was compared to the S. cerevisiae protein in its ability to reduce protein haze, and it was shown that the P. pastoris protein could reduce haze, but not as effectively as the S. cerevisiae protein. The finding that Hpf2 does not affect the sensory properties of wine is essential if Hpf2 is to be used commercially, as winemakers and wine consumers would most likely reject an additive that alters the wine aroma or palate. This work has brought the wine industry a step closer to a new method for protein haze prevention in white wines. / Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 2008

White wine clarity is essential, but it can be marred by the presence of a protein haze. This protein haze is predominantly formed by grape-derived thaumatin-like proteins and chitinases, which can slowly denature and aggregate if left in bottled wines. Currently bentonite fining is used by the wine industry to prevent protein haze. Bentonite consists of fine clay particles that, when added to wine, bind and remove the haze-forming proteins. However this method is inconvenient, time-consuming, and causes significant losses of wine. It is estimated that this process costs the Australian wine industry $50 m annually in wine losses alone. Alternatives are thus being investigated. The principal objective of this thesis was to investigate the sensory effects on wine of an alternative method to bentonite fining: addition of haze protective factor 2, known as Hpf2. Hpf2 is a Saccharomyces cerevisiae mannoprotein that has been shown to reduce protein haze in wines. It is a highly mannosylated 180 kDa protein, of which approximately 75% by weight is mannose. Previous work has shown that the addition of approximately 200 mg L⁻¹ Hpf2 to wines reduces the visible haze in wine by approximately 50%. Hpf2 is naturally present in wines at concentrations of less than 10 ng L⁻¹, much lower than the concentration required for haze protection activity. However, the sensory impacts involved with the addition of such high concentrations of Hpf2 in wine have never been studied. This knowledge is essential for the future commercial prospects of this alternative approach to protein stabilisation of wine. To undertake sensory studies, over 1 g of Hpf2 would be required. Presently, the laboratory-scale process for the production of a 6-histidine tagged version of the protein, 6xHisHpf2, in a laboratory yeast strain of S. cerevisiae, produces only milligram quantities. Consequently, the first challenge of this research was to scale up the existing process to produce sufficient quantities of Hpf2. The first attempt to increase the production level was by over-expression in the bacteria Escherichia coli. Although several approaches were trialled, 6xHisHpf2 was unable to be successfully and consistently expressed in this system. The second method was by improving the original yeast expression system, and the expression level was able to be improved approximately 10-fold. This improved expression method was scaled up to produce and then purify over 1 g of protein. Several quantification methods were assessed to determine the efficiencies of each purification step, with slot blot analysis proving successful. Sensory trials were conducted to establish the effect of 6xHisHpf2 on wines, with duo-trio studies conducted assessing both aroma and palate of the wines. Invertase, another yeast haze protective factor, was also trialled. It was found that the addition of an active level of 6xHisHpf2 or invertase did not cause a significant difference in the aroma or palate of wines. In addition to this main study, the role of the glycosylation was studied. 6xHisHpf2, produced in a different yeast, Pichia pastoris, was found to be 83 kDa, with only 50% mannose. This protein was compared to the S. cerevisiae protein in its ability to reduce protein haze, and it was shown that the P. pastoris protein could reduce haze, but not as effectively as the S. cerevisiae protein. The finding that Hpf2 does not affect the sensory properties of wine is essential if Hpf2 is to be used commercially, as winemakers and wine consumers would most likely reject an additive that alters the wine aroma or palate. This work has brought the wine industry a step closer to a new method for protein haze prevention in white wines. / Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 2008