Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder which is caused by a mutation in the gene encoding acid β-glucocerebrosidase (GBA, EC 3.2.1.45). Deficient activity in GBA leads to a wide variety of clinical phenotypes, including visceral symptoms such as hepatospenomegaly as well as neurological symptoms. Current enzyme replacement therapy is effective in treating visceral symptoms but cannot cross the blood-brain barrier to target neurological manifestations. Another drawback to current therapy is the high cost to patients due to present protein expression strategies. Recently, protein transduction domains, such as the synthetic PTD4 domain, have been proposed as a therapeutic strategy for drug delivery to the central nervous system. In the present study, we use an economical yeast expression system, Pichia pastoris, to produce a recombinant fusion protein GBA-PTD4, and semi-preparative hydrophobic interaction chromatography and gel filtration chromatography for purification. Results show that final preparations are near homogenous, with GBA-PTD4 accounting for approximately 76% of total protein and only one major contaminant. A cell line expressing GBA without a transduction domain was also created in anticipation of further cellular uptake studies. Future research will focus on large scale enzyme expression in fermentation systems and more direct purification methods such as immunoaffinity chromatography for better protein recovery. / Graduate

Student Number : 0213014A - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cytosolic glutathione transferases (GSTs) are obligate stable homo- and heterodimers comprising two GST subunits. Interactions across the subunit interface play an important role in stabilising the subunit tertiary structure and maintain the dimeric structure required for activity. The crystal structure of a rat Mu class GST consisting of two type one subunits (rGST M1-1) reveals a lock-and-key motif and a mixedcharge cluster at the subunit interface. Previous investigations revealed the lock-andkey motif was not essential for dimerisation. It was therefore postulated that the mixed-charge cluster at the dimer interface is primarily responsible for subunit association. Statistical analyses of individual rGST M1-1 chains did not predict the presence of any charge clusters. This suggests that the mixed-charge cluster forms only upon dimerisation and reinforces the probability that quaternary structure stabilisation is a major role of the mixed-charge cluster. Arginine 81 (Arg-81), a structurally conserved residue in the GST family involved in the mixed-charge cluster, was mutated to alanine. Phenylalanine 56 (Phe-56), the ‘key’ residue in the lock-and-key motif, was mutated to serine. These changes were engineered to disrupt the mixed-charge cluster and the lock-and-key motif situated at the dimer interface of rGST M1-1. Sizing by gel filtration chromatography of the mutant GST identified that these engineered amino acids resulted in a stable monomeric protein (F56S/R81A rGST M1). The F56S/R81A rGST M1 displayed almost no catalytic activity, suggesting perturbations of the active site or substrate binding sites. Structural investigations of the monomer by far- and near-UV circular dichroism revealed a similar secondary structural content to the wild-type. However, the tryptophan fluorescence properties suggested the tryptophans were situated in more hydrophilic environments than in the wild-type. ANS binding studies indicated a large increase in the accessible hydrophobic surface area of the monomer. Ureainduced equilibrium unfolding of F56S/R81A rGST M1 follows a cooperative twostate unfolding model. The unfolding data indicates decreased conformational stability and a large increase in the solvent exposed surface area of the monomer. In conclusion, the mixed-charge cluster at the dimer interface of rGST M1-1 is essential for monomeric association, which subsequently contributes to catalytic activity of the dimer and the stabilities of individual rGST M1-1 subunits.

<p><em>Size exclusion chromatography</em> (SEC) was applied for purification of psychoactive biomolecules from plants. These molecules are in the same molecular weight range, but do not necessarily share other chemical properties, that makes the SEC technique efficient. By applying SEC as a first purification step much of the co-extractives from the plants can easily be removed. Large amounts of target substance can be obtained with little effort if the system is automated. Combining SEC with a second purification step, consisting of normal phase chromatography, provides high purity of the target substance.</p><p>Both known and unknown psychoactive biomolecules can easily be purified using the purification method developed in this Master's Thesis. Purifications that previously required long time and much "hands-on" can be completed much faster and with less manual work.</p><p>The method developed was tested on cannabis, coffee and 'Spice' with good results.</p>

Size exclusion chromatography (SEC) was applied for purification of psychoactive biomolecules from plants. These molecules are in the same molecular weight range, but do not necessarily share other chemical properties, that makes the SEC technique efficient. By applying SEC as a first purification step much of the co-extractives from the plants can easily be removed. Large amounts of target substance can be obtained with little effort if the system is automated. Combining SEC with a second purification step, consisting of normal phase chromatography, provides high purity of the target substance. Both known and unknown psychoactive biomolecules can easily be purified using the purification method developed in this Master's Thesis. Purifications that previously required long time and much "hands-on" can be completed much faster and with less manual work. The method developed was tested on cannabis, coffee and 'Spice' with good results.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

Newcastle disease virus (NDV) is one of the paramyxovirus that has been studied at length since this virus infects the birds of all species. NDV is highly virulent in chickens and results in a high mortality rate because the V protein of NDV is found to inhibit the avian immune response system. No drugs are available for treating NDV therefore, determining the structure of V protein would help in developing a drug that can inactivate the V protein, thereby increasing the host immune response against viral infection. The research here is focused on purification and initial structural analysis of the V protein of NDV. The V protein was purified by gel filtration chromatography and the structure was studied using fluorescence and CD spectroscopy, and NMR. The results suggested that the V protein is unstructured. The research also involved purification and structural analysis of the flowering locus T (FT) protein, which is found to play a major role in theninitiation of flowering in plants. Structural analysis of the FT protein may help in finding the possible domains of the FT protein that interacts with other plant proteins, leading ton flowering. The FT protein was purified by ion exchange chromatography and the structure was studied by fluorescence and CD spectroscopy. The fluorescence data suggested that the FT protein may be folded, where as the CD data was inconclusive. More accurate secondary structure information about the protein could be obtained using NMR, but since the concentration of the FT protein was too low (0.007 mM), proper NMR study was not possible.

Bacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.

Bacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.

Made available in DSpace on 2017-07-10T17:37:44Z (GMT). No. of bitstreams: 1 Cristiane_Claudia_Meinerz.pdf: 977726 bytes, checksum: 47602589d307d4398cc6061bcd0d1eeb (MD5) Previous issue date: 2010-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Induction of resistance involves the activation of plant defense mechanisms in response to treatment with biotic or abiotic elicitors. The application of plant extracts in order to induce resistance mechanisms is an interesting alternative to chemical control, however, besides the presence of inducers, can occur the presence of suppressors. This study aimed to partially purificate through gel filtration chromatography (GFC) and precipitation with ammonium sulfate (SA), compounds present in decoct of Adiantum capillus-veneris, capable to induce defense mechanisms in sorghum mesocotyls, including phytoalexins and peroxidase, polyphenoloxidase (PPO), phenylalanine ammonia-lyase (PAL) and chitinase. The decoct 1% was fractionated with concentrations of ammonium sulfate, 0-20%, 20-40%, 40-60%, 60-80% and 80-100% of SA and those fractions were subjected to GFC. We obtained nine protein peaks and one glucosic peak for decoct with molecular weights ranging from 0.61 to 0.01 KDa; to fraction 0-20% were obtained two protein and two glucosic peaks, with molecular weights lower than 0.01 KDa, and concentration of sugars ranging from 4.1 to 17.5 mg mL-1; to fraction 20-40% were obtained three protein peaks (0.98 to 111.5 KDa) and five glucosic peaks (11.3 to 73.7 mg mL-1); to fraction 40-60% were obtained two protein peaks (0.09 to 111.5 KDa) and two glucosic peaks (5.6 to 7.5 mg mL-1); to fraction 60-80% were obtained six protein peaks (lower than 0.02 KDa) and two glucosic peaks (16.5 to 51.3 mg mL-1); and to fraction 80-100% were obtained three protein peaks (lower than 0.09 KDa). Sorghum mesocotyl were treated with fractions from the GFC, and decoct, acibenzolar-S-methyl (ASM) (125 mg L-1 of a. i. as elicitor of reference) and sodium phosphate buffer 10 mM pH 6.0. After incubation of 96 h were measured the levels of phytoalexins in mesocotyls and the activity of defense-related enzymes in leaves. Treatment with peak II (0,09 KDa) induced phytoalexin 6.68% more than. Among the fractionn, 60-80% increased 76% compared to ASM. To peroxidase the peak IV (lower than 0,01 KDa) increased 21% the activity compared to control water, and 44% compared to ASM. For the fraction 0-20% the protein peak II (lower than 0,01 KDa) increased 39% the activity in relation to the fraction 0-20% and 19% in relation to decoct. The fraction, 80-100% increased 89% compared to, ASM. For the PPO the peak VI (lower than 0,01 KDa) from decoct decreased 88% the activity compared to ASM. For PAL the peak II (lower than 0,01 KDa) from fraction 0-20% was 91% higher than decoct. For chitinase 1% peak IV (lower than 0,01 KDa) from decoct was 68% higher than the ASM. It was possible to induce defense mechanisms in sorghum by the application of partially purified fractions from A. capillus-veneris, which can allow to obtain new molecules and development alternative methods to control plant diseases / A indução de resistência envolve a ativação de mecanismos de defesa latentes existentes nas plantas em resposta ao tratamento com agentes bióticos ou abióticos. A aplicação de extratos vegetais visando à indução de mecanismos de resistência é uma alternativa interessante ao controle químico, entretanto, nestes extratos pode ocorrer além da presença de indutores, a presença de supressores. Este trabalho teve por objetivo a purificação parcial, por meio de cromatografia de filtração em gel (CFG) e precipitação com sulfato de amônio (SA), de compostos presentes em decocto de avenca (Adiantum capillus-veneris), eficientes na indução de mecanismos de defesa em mesocótilos de sorgo, incluindo as fitoalexina deoxiantocianidinas e as proteínas peroxidase, polifenoloxidase, fenilalanina amônia-liase e quitinase, buscando selecionar frações potencialmente eficientes na indução de resistência em plantas. Decocto (EA 1%) de A. capillus-veneris foi fracionado com concentrações de sulfato de amônio de 0-20%, 20-40%, 40-60%, 60-80% e 80-100% e esses cortes foram submetidos à cromatografia de filtração em gel (CFG). Foram obtidos nove picos protéicos e um pico glicídico para EA 1% com massas moleculares variando de 0,61 à 0,01 KDa; no corte 0-20% foram obtidos dois picos protéicos e dois glicídicos, com massas moleculares menores que 0,01 KDa, e concentração de açúcares redutores variando de 4,1 a 17,5 µg mL-1; no corte 20-40% três picos protéicos (111,5 à 0,98 KDa) e cinco glicídicos (11,3 a 73,7 µg mL-1 de açúcares); no corte 40-60% dois picos protéicos (111,5 à 0,09 KDa) e dois glicídicos (5,6 a 7,5 µg mL-1); no corte 60-80% seis picos protéicos (menor que 0,02 KDa) e dois glicídicos (16,5 a 51,3 µg mL-1); e no corte 80-100% três picos protéicos (menor que 0,09 KDa). Mesocótilos de sorgo foram tratados com as frações provenientes da CFG, além do decocto a 1%, acibenzolar-S-metil (ASM) (125 mg. L-1 do i.a. como elicitor de referência) e tampão fosfato de sódio 10 mM pH 6,0, totalizando 42 tratamentos. Após incubação por um período de 96 h, avaliou-se dos teores de fitoalexinas nos mesocótilos e análises bioquímicas dos folíolos. O tratamento pico II (0,09 KDa) do EA 1% mostrou-se eficiente na indução de fitoalexinas, sendo superior em 6,68% ao ASM. Entre os cortes, 60-80% permitiu incremento de 76% em relação ao ASM. Para peroxidase o pico IV (menor que 0,01 KDa) do EA 1% incrementou 21% a atividade em relação a testemunha água e 44% ao ASM. Para os precipitados 0-20% o pico protéico II (menor que 0,01 KDa) promoveu incremento de 39% na atividade em relação ao corte 0-20% e 19% para o EA 1%. O precipitado 80-100% foi superior 89% ao ASM. Para polifenoloxidase o pico protéico VI (menor que 0,01 KDa) do EA1% reduziu 88% a atividade em relação ao ASM. Para fenilalanina amônia-liase o pico protéico II (menor que 0,01 KDa) do corte 0-20% foi 91% superior ao EA 1%. Para quitinase o pico protéico IV (menor que 0,01 KDa) do EA 1% foi 68% superior ao ASM. Foi possível induzir mecanismos de defesa em sorgo pela aplicação de frações parcialmente purificadas de A. capillus-veneris, o que pode permitir a obtenção de novas moléculas e o desenvolvimento de métodos alternativos para controle de doenças em plantas