Human α-N-acetylglucosaminidase (Naglu) is a lysosomal enzyme implicated in the rare metabolic storage disorder Mucopolysaccharidosis III type B (MPS IIIB). A deficiency in Naglu results in a buildup of heparan sulfate in lysosomes, which is most detrimental in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is currently ineffective in treating the neurological symptoms of MPS IIIB due to the inability of Naglu to cross the blood-brain barrier. This laboratory uses a Spodoptera frugiperda insect cell system to express recombinant Naglu conjugated to a synthetic protein transduction domain with the intent to allow Naglu to cross the blood-brain barrier and treat the neurological symptoms. In the present study, we aimed to purify a recombinant Naglu-PTD4 fusion protein in order to assess its capacity to cross cellular membranes. A three-step method involving multi-modal, hydrophobic interaction, and gel filtration chromatography was optimized to achieve pure Naglu-PTD4, in good yield. Cellular uptake by human MPSIIIB fibroblasts of Naglu-PTD4 was not detectable. It is hypothesized that additional amino acids, including a hexahistidine domain, following the PTD4 domain limited the fusion protein’s membrane transduction capacity. Future studies will focus on removing the additional amino acids and adjusting the purification method as necessary. The ultimate goal of this research is to develop a large-scale recombinant Naglu production protocol for enzyme replacement therapy of MPS IIIB. / Graduate

A síntese ribossomal é uma das maiores atividades em células eucarióticas. Este processo inicia-se no nucléolo e é finalizado após a exportação das subunidades 40S e 60S para o citoplasma. Três dos RNAs ribossomais de eucariotos (18S, 5.8S e 25S) são sintetizados como um transcrito primário de 35S, o qual é processado através de uma complexa e ordenada série de modificações nucleotídicas e clivagens endo e exonucleolíticas. Estas reações dependem de aproximadamente 170 proteínas, 80 small nucleolar RNAs e de seqüências no pré-rRNA. Os fatores trans-atuantes envolvidos no processamento podem ser agrupados como RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) e exonucleases, que incluem o complexo exossomo. O exossomo de levedura é formado por 10 proteínas essenciais que atuam na maturação de rRNAs, snRNAs, snoRNAs, além da degradação de mRNAs incorretamente processados. A estrutura do exossomo de archaea foi descrita recentemente, mas ainda não existem muitas informações sobre a regulação deste complexo e sobre a participação de cofatores que interagem de forma transiente com o exossomo. Diante disso, este trabalho visou a caracterização funcional das proteínas que formam o anel de RNases PH em Saccharomyces cerevisiae, assim como a caracterização estrutural e funcional de possíveis cofatores do exossomo de Saccharomyces cerevisiae, Nop17p e Ylr022p, e do exossomo de Pyrococcus, Pab418p, Pab1135p e aNip7p. Os dados obtidos evidenciam que a atividade exonucleolítica do exossomo de levedura, assim como o de archaea, é dependente da formação de heterodímeros; Ylr022p, uma proteína de levedura com função não caracterizada, liga inespecificamente RNA in vitro, mas mais eficientemente alguns RNAs in vivo. Dentre as proteínas de archaea, Pab418p e aNip7p também ligam RNA, e como demonstrado aqui, aNip7p influencia significativamente a atividade do exossomo de archaea. / The synthesis of ribosomes is one of the major metabolic pathways in eukaryotic cells. This process starts in the nucleolus and ends with the export and final maturation of the ribosomal subunits 40S and 60S in the cytoplasm. Three eukaryotic ribosomal RNAs (18S, 5.8S and 25S) are synthesized as a 35S primary transcript (35S pre-rRNA), which is then processed by a complex and ordered series of nucleotide modifications and endo- and exonucleolytic cleavage reactions. These processing reactions depend on 170 proteins, 80 small nucleolar RNAs and specific pre-rRNA sequences. The trans-acting factors, that take part in the processing can be grouped as RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) and exonucleases, including the exosome. The yeast exosome is composed of 10 essential proteins that function in the processing of rRNAs, snRNAs, snoRNAs and in the degradation of aberrant mRNAs. Recently, the archaeal exosome structure was determined, but no information is yet available on the regulation of the exosome function or on the possible role of the cofactors that transiently interact with it. The main goals of this work were the functional characterization of the protein components of the Saccharomyces cerevisiae exosome RNase PH ring, as well as the structural and functional characterization of the possible cofactors of that complex, Nop17p and Ylr022p. Since the recent characterization of the Pyrococcus exosome, the study of the archaeal exosome cofactors, Pab418p, Pab1135p and aNip7p, was also included in this work, in order to correlate the data on the complex of these different organisms. Our results show that the exonucleolytic activity of the yeast exosome is dependent on the heterodimers formation, as described for archaea. Although it is not clear how Nip7p affects the exosome function in yeast, aNip7p binds RNA and inhibits a-exosome activity in vitro. Yeast Ylr022p binds RNA inespecificaly in vitro, but coprecipitates specific RNAs more efficiently from total cell extracts. Its archaeal orthologue, Pab418p, also binds RNA, but does not affect significantly a-exosome function.

O desenvolvimento de vacinas contra o vírus do papiloma humano (HPV) representa uma importante alternativa para o controle da infecção sexualmente transmissível e do câncer cervical. Neste trabalho exploramos uma estratégia vacinal inédita contra tumores induzidos pelo HPV-16 empregando a proteína E7 obtida após fusão genética com a glicoproteína D do vírus herpes tipo 1, uma proteína com propriedades adjuvantes para linfócitos T. A proteína recombinante gDE7 foi expressa em bactérias e em células de inseto e purificada por cromatografia de afinidade. A proteína gerada em bactérias foi administrada nas formas insolúvel e solúvel como vacina em camundongos. A gDE7 insolúvel conferiu 80% de proteção profilática para o crescimento tumoral, a gDE7 que manteve a forma solúvel após refolding protegeu 100% dos animais nas mesmas condições. Já a proteção terapêutica alcançou 30% dos animais. Além disso, verificamos o potencial neutralizante dos soros gerados frente ao HSV-1. As condições de obtenção das proteínas expressas em células de inseto também foram estabelecidas. / The development of vaccines against human papillomavirus (HPV) represents an important alternative to control the sexually transmitted infection and cervical cancer. In this paper we explored a novel vaccine strategy against tumors induced by HPV-16 using the E7 protein obtained after genetic fusion to the glycoprotein D of herpes virus type 1, a protein with adjuvant properties for T lymphocytes. The recombinant protein gDE7 was expressed in bacteria and in insect cells and purified by affinity chromatography. The protein generated in bacteria was administered in soluble and insoluble forms as a vaccine in mice. The insoluble gDE7 gave 80% prophylactic protection for tumor growth, the gDE7 that kept the soluble form after refolding protected 100% of the animals under the same conditions. The therapeutic protection reached 30% of the animals. We also verified the neutralizing potential of sera generated against the HSV-1. The conditions for obtaining the proteins expressed in insect cells were also established.

Expanded bed chromatography using ion-exchange media is a powerful first step in purification processes. Expanded bed chromatography can be used to extract components from complex and viscous solution. This can be achieved because of the void created between adsorbent particles where as in packed bed chromatography, the adsorbent is too compact and dense for a complex feed stock to flow through. Expanded bed chromatography was used to purify bovine serum albumin (BSA) from chicken egg white (CEW). The high viscosity of CEW presents a unique challenge for efficient large-scale protein purification. This project aimed to optimize and evaluate a separation method that is believed to be particularly suitable for high viscosity solutions: expanded-bed ion exchange chromatography. The BSA was admixed into the CEW and the solution was pumped through the column for purification. The media used in the column was Streamline DEAE which is an anion-exchanger. The yield obtained was 85% and the purity was 57%. A mathematical model to understand and predict the behavior of expanded bed chromatography was developed to provide an estimation of the breakthrough curves obtained for BSA. A small sized porous dense adsorbent was also synthesized to enhance the purification process. This zirconia-based adsorbent allows use of higher flow velocities that is a key factor when working with viscous fluids such as chicken egg white.

STRAP (Stress responsive activator of p300) is a 440 amino acid protein, predicted to have 6 TPR (Tetra-Tri-Co-Peptide Repeats) motifs, known to mediate protein-protein interactions. STRAP has been shown to form a complex with proteins p300 and JMY (Junctional Mediatory Protein), and is implicated in the DNA damage, heat shock response pathway, regulation of the Glucocorticoid receptor and in the function of p53.The aims of this project were to clone, express and purify full length and truncated human STRAP (hSTRAP) variants in high quantities. Full length and shorter hSTRAP fragments, which contain different combinations of the predicted TPR motifs and hence cover different regions, would be then structurally characterised by various structural and biophysical experiments. Another important aim was to identify interacting partners of hSTRAP in breast cancer and to map the position of their interaction sites to different parts of the protein. To this direction GST- and His- tagged full length hSTRAP, as well as His- tagged truncated hSTRAP protein variants have been successfully cloned, expressed and purified. Independent and reproducible biochemical pull-down assays have been carried out in MCF7 breast cancer cells, followed by mass spectrometry-based proteomics analysis which identified 25 hSTRAP-interacting partners from various signaling pathways such as regulation of the actin cytoskeleton and translation. In addition, crystallization trials were carried out using pure His-hSTRAP(1-440) protein, which were unfortunately un-successful. Various hSTRAP protein variants have been characterized by CD, showing that hSTRAP(1-150), His-hSTRAP(1-440), hSTRAP(1-219), hSTRAP(151-284) and hSTRAP(285-440) comprise of alpha and β structures, but the hSTRAP protein variants show no clear cooperative unfolding transitions, suggestive of molten globule states. NMR on hSTRAP(1-219), hSTRAP(1-150) and hSTRAP(151-284) have shown these proteins are not folded at a tertiary structure level. We conclude that a protocol has been established to clone, express and purify various hSTRAP variants and the thermal and secondary structure characteristics of each have been determined, although the 3D structure could not be solved. Pull-down assays followed by proteomic analysis have shown that hSTRAP is implicated in many aspects of cellular regulation.

DNA in cells is constantly damaged by both endogenous and exogenous genotoxic agents. DNA repair is a cellular machinery that counters DNA damage and thus preserve genome integrity. Nucleotide excision repair (NER) in Escherichia coli (E. coli) is one of the DNA repair systems that recognizes and removes a variety of DNA damage such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand cross-links, etc. The genes responsible for E. coli NER incisions are UvrA, UvrB, and UvrC. As the first step of E. coli NER, DNA damage recognition is achieved through the UvrA2B complex. Purification of UvrA, UvrB, and UvrC is essential for research to understand the molecular mechanisms of NER and carcinogenesis. Although UvrA, a 115 kDa protein, has been successfully purified in our lab in the past, the experimental procedures were very time-consuming and technically challenging. In this study we employed IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system to subclone the cDNA of UvrA and express and purify the recombinant UvrA protein by a single-column step using the cloned expression construct. Furthermore, the purified protein was found to be fully functional in the UvrABC incision assay in which the DNA adduct of FABP [N-(20-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl] was efficiently cleaved in a time course-dependent manner.

<p>The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in <em>Escherichia coli</em>, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys₂₇₈-Cys₄₄₃being the most important and Cys₂₁₆-Cys₄₂₀ and Cys₃₃₀-Cys₃₈₀ being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.</p>

To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.   In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.   The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors. / QC 20101102

The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in Escherichia coli, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys278-Cys443 being the most important and Cys216-Cys420 and Cys330-Cys380 being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.

Heparin binding (HB) proteins mediates a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins could bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to the currently available methods. One of the most important classes of heparin binding protein is the fibroblast growth factors (FGFs) and its receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which exhibit several disadvantages, including time-consuming experimental procedures and regeneration and results in high cost for production of recombinant proteins. Authenticity of the purified proteins was verified by SDS-PAGE and MALDI mass spectrum analysis. Results of the heparin binding chromatography and steady state fluorescence experiments showed that the FGF-1 and the D2 are in a native biologically active conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.