Chow Ka-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 52-55). / Abstracts in English and Chinese. / Chapter 1 --- Introduction / Chapter 1.1 --- Chemical sensors --- p.1 / Chapter 1.2 --- Quartz crystal microbalance --- p.4 / Chapter 1.3 --- Concept of affinity mass sensor --- p.8 / Chapter 1.4 --- Film immobilization technologies --- p.9 / Chapter 1.5 --- Research outlines --- p.13 / Chapter 2 --- Experimental / Chapter 2.1 --- Sensor fabrication --- p.14 / Chapter 2.2 --- Flow-through cell --- p.16 / Chapter 2.3 --- Analysis procedures --- p.19 / Chapter 2.4 --- Response curve --- p.19 / Chapter 2.5 --- Experimental setup --- p.21 / Chapter 3 --- Detection of ascorbic acid by affinity mass sensor based on 3-aminophenylboronic acid / Chapter 3.1 --- Conventional analytical methods --- p.23 / Chapter 3.2 --- Research method - affinity mass sensor based on APBA --- p.24 / Chapter 3.3 --- To locate the binding site in ascorbic acid --- p.25 / Chapter 3.3.1 --- Steric energy calculated by molecular modeling --- p.26 / Chapter 3.4 --- Optimization of experimental variables --- p.29 / Chapter 3.4.1 --- Effect of pH --- p.29 / Chapter 3.4.2 --- Effect of sample volume --- p.30 / Chapter 3.4.3 --- Effect of flow velocity --- p.30 / Chapter 3.5 --- Calibration and Reproducibility --- p.32 / Chapter 3.6 --- Kinetic analysis --- p.33 / Chapter 3.7 --- Stability of sensor --- p.37 / Chapter 3.8 --- Interference studies --- p.37 / Chapter 3.9 --- Determination of ascorbic acid in real samples --- p.39 / Chapter 3.9.1 --- Results and Discussion --- p.39 / Chapter 3.10 --- Comparison with conventional ascorbic acid sensors --- p.42 / Chapter 3.11 --- Summary --- p.42 / Chapter 4 --- Boronic acid derivatives for the detection of sugars / Chapter 4.1 --- Scope of this work --- p.43 / Chapter 4.2 --- Results and Discussion --- p.44 / Chapter 4.3 --- Summary --- p.49 / Conclusion --- p.50 / References --- p.52 / List for tables --- p.56 / List for figures --- p.57 / Appendix I --- p.59 / Appendix II --- p.61

A Gonadotrofina Coriônica Eqüina (eCG) é produzida pela égua prenhe e tem ação folículo estimulante e luteinizante em animais domésticos não eqüídeos. Um pool formado por plasma de 4 éguas prenhes, com média de 69 dias de gestação, foi purificado em coluna cromatográfica com resina de afinidade Blue Sepharose FF (BS). As frações que adsorveram à resina BS foram purificadas em coluna cromatográfica com resina de afinidade Concanavalina A 4B (ConA). As frações que não adsorveram à resina BS também foram purificadas em coluna cromatográfica com resina de afinidade ConA. O mesmo pool de palsma foi diafiltrado, em cartucho de hemodiálise. O diafiltrado foi aplicado em coluna cromatográfica com resina de afinidade ConA. Atividade biológica (UI/mL) do plasma, do diafiltrado e das frações purificadas foram quantificadas por ensaio biológico com ratas impúbres. As atividades biológicas encontradas no plasma e no plasma diafiltrado foram de 3,63 e 5,14UI/mL, respectivamente. A atividade biológica encontrada nas frações que adsorveram à BS foi de 3,50UI/mL. Não foi encontrarda atividade biológica nas frações que não adsorveram à BS. A atividade biológica contida nas frações que adsorveram à BS e que também adsorveram a ConA foi de 3,65UI/mL. O rendimento do processo cromatográfico onde o plasma foi adsorvido pela BS e pela ConA, foi de 69,52%. Não foi encontrada atividade biológica nas frações obtidas da aplicação do plasma diafiltrado em coluna de ConA. O processo cromatográfico com uso de BS seguido de ConA mostou-se eficaz em purificar a eCG do plasma de éguas prenhes. / The equine Chorionic Gonadotrophin (eCG) is produced by the pregnant mare and has follicle-stimulant and luteinizing actions on non-equine domestic animals. A pool formed by the plasma of 4 pregnant mares (with mean gestation of 69 days) was purified in chromatographic column with Blue-Sepharose FF affinity resin (BS resin). Fractions adsorbed by BS resin were then purified in chromatographic column with Concavalin A 4B affinity resin (ConA resin). The fractions not adsorbed by the BS resin were also purified in chromatographic column with ConA resin. The same plasma pool was dialyzed in hemodialysis cartridge. The dialyzed was applied in chromatographic column with ConA resin. Biological activities (in IU/mL) of the plasma, of the dialyzed and of the purified fractions were quantified in a biological assay with female rats that did not reach puberty. The biological activities found in the plasma and dialyzed were of 3.63 and 5.14 IU/mL, respectively. Fractions that were adsorbed by BS had a biological activity of 3.50 IU/mL. No biological activity was found in fractions that were not adsorbed by BS. Biological activity found in fractions adsorbed by both BS and ConA was of 3.65 IU/mL. When plasma was both adsorbed by BS and ConA, the chromatographic process yield had results of 69.52%. No biological activity was found in the fractions obtained from the administration of dialyzed plasma in ConA column. The BS - followed by ConA -chromatographic process showed efficacy in purifying the eCG from the plasma of pregnant mares.

Therapeutic antibodies dominate the pharmaceutical market and improve the lives of millions of people annually. One important step when developing new medicines is to kinetically characterise the drug candidates. For antibodies this is difficult since many antibody reactions are extremely slow. By combining a mathematical formula that was recently published with the well-established technology from Gyros Protein Technologies, a new method for full kinetic characterization was developed and tested in this master thesis. The method provided precise data for five antibodies while also proving to be highly reproducible. By using small sample volumes, unlabelled reagents and having the reaction proceed in solution, this method offers advantages compared to many conventional approaches.

Les immunoessais en phase solide sont utilisés pour le diagnostic in vitro afin de détecter ou de quantifier une molécule dans un échantillon biologique. Ils s'appuient sur l'interaction spécifique entre un antigène et un anticorps. Habituellement, des anticorps spécifiques aux antigènes à détecter sont immobilisés sur une surface solide pour capturer les antigènes d'intérêt et les séparer du reste de l'échantillon.Lors du développement d'un immunoessai, la sensibilité, la spécificité et le temps d’analyse sont optimisés par le choix - classiquement empirique - de ligands, de supports solides, de débits,… Une meilleure compréhension et prédiction des interactions moléculaires complexes se produisant au cours d’un immunoessai seraient utiles pour : identifier les paramètres critiques des immunoessais, simplifier et accélérer le processus d’identification des meilleures conditions opératoires et améliorer les immunoessais existants.L'instrument VIDAS®, commercialisé par bioMérieux, est l'un des systèmes d’immunoessais les plus utilisés dans les laboratoires cliniques. Dans ce travail de thèse, deux outils expérimentaux basés sur la chromatographie inverse sont construits et testés. Un modèle prédictif de la cinétique d'interaction anticorps/antigène est développé. Les outils expérimentaux, fonctionnant dans des conditions très proches du VIDAS®, sont utilisés pour valider le modèle et estimer ses paramètres caractérisant les interactions anticorps/antigène à partir de courbes expérimentales. Dans l’avenir et à partir des résultats, un des outils expérimentaux associé au modèle pourra être utilisé par bioMérieux pour concevoir des systèmes d’immunoessais / Solid-phase immunoassays are used for in vitro diagnostic to detect the presence or measure the concentration of a molecule of interest in a biological sample. They rely on the specific interaction between an antigen and an antibody. Usually, antibodies specific to the antigens to be detected are immobilized on a solid surface to capture the antigens of interest and separate them from the rest of the sample components. During solid-phase immunoassay development, sensitivity, specificity and time-to-result need to be optimized through the choice of dedicated ligands, solid supports, flow rates,… Classically, these choices are made empirically. A better understanding and prediction of the complex molecular interactions that occur in the different steps of a diagnostic immunoassay is likely to be useful to: identify the critical parameters of immunoassays, simplify and speed-up the process of identification of the best immunoassay conditions and improve the immunoassays currently available. The VIDAS® instrument, commercialized by bioMérieux is one of the most widely used immunoassay system in clinical laboratories worldwide. In this PhD work, two experimental tools based on inverse chromatography are built and tested. A predictive model of antibody/antigen interaction kinetics in immunoassays is developed. The experimental tools which mimic VIDAS® process conditions are used to validate the predictive model and to estimate model parameters characterizing antibody/antigen interaction kinetics from experimental curves. In the future, based on the results, one of the experimental tools associated with the model could be used by bioMérieux to design immunoassay systems

Malaria is a major threat to the public health worldwide as it is affecting populations in tropical and subtropical areas globally. Among those populations are around 40% of pregnant women and children who are susceptible to this disease. Plasmodium falciparum is the most lethal agent that causes malaria in human. Currently, there is drug resistance against antimalarial drugs in parasite against treatment of malaria infections, it is essential to search for new drug targets in order to find cure and alleviate suffering of human population. There are approximately 100 protein kinases in P. falciparum that are involved in phosphorylation of asexual blood stage. Hence, the phosphorylation plays an important part in the development of different stages of malarial parasites. Due to their significance in the parasite life cycle, one of the protein kinase of P. falciparum belongs to the ABC-1 family of proteins. PfABCK2 can be a therapeutic target due to its higher expression during the late schizont stage of blood stage form. The bioinformatic analysis and preliminary results of PfABCK2 showed the heterologous expression of this protein. Hence, the gene of PfABCk2 was ligated into pET21a+ vector with His-tag at C-terminus and transformed into BL-21 (DE3) competent cells that were verified through Miniprep and DNA sequencing. Furthermore, this gene construct is utilized to heterologous express this protein with IPTG and afterwards purified the recombinant protein kinase using nickel affinity chromatography as shown on 10% SDS-PAGE with the expected 36 kDa protein band. Therefore, the aim of this study is to partially characterize PfABCK2 protein kinase utilizing molecular cloning, heterologous express and protein kinase activity assay.

<p>Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. </p><p>A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. </p><p>Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTs^TM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. </p><p>The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.</p>

This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting. In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines. In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTsTM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography. / <p>De två (2) första författarna delar förstaförfattarskapet.</p>

Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity chromatography followed by liquid chromatography mass spectrometry from HEK-293 cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP), ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP), phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were selected for interaction studies. Immunofluorescence analysis showed that CHERP co-localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293 cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in CHERP and FHL1 which may represent novel regulatory mechanisms involved in excitation-contraction coupling.