A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...

The MS-818 compound is used in the proliferation process of neuronal cells and many biological activities that accompany this process such as astrocyte differentiation, inhibition of neuronal apoptosis, and fraction repairs. We do know the effects of this compound, but the mechanism of action remained uncertain until now. To determine the pathway of this compound, NT2 cells were cultured and lysed to isolate the proteins. Affinity Chromatography was performed in order to immobilize the MS-818 compound to a Hi-Trap NHS column. The NT2 protein sample was injected through the column and eluted with a MS-818 concentrated, high salt content elution buffer. SDS-PAGE was then performed to isolate the proteins that bound to MS-818. The gel was visualized using Coomassie Blue. The results indicate that there are two proteins associated in the mechanism of this compound. A standard protein marker ranging from 10 kDa to 250 kDa was used to compare the bands. The findings indicate that one of the protein bands is slightly less than 250kDa and the other is between 50-75 kDa. When the proteins are confirmed by mass spectrometry sequencing, this will help to promote this compound as a drug candidate.

No description available.

No description available.

This thesis describes the use of functional nucleic acid (FNA) superstructures entrapped within monolithic macroporous sol–gel-derived silica for solid-phase flow-based sensing of small molecules and macromolecular proteins. The work described herein overcomes a long-standing issue with entrapment of biomolecule into sol–gel-derived materials; the mesoporous pore morphology required to retain entrapped biomolecules prevents detection of large analytes as these can’t access the entrapped species. It is shown that large DNA superstructures can be produced through rolling circle amplification of a functional nucleic acid, resulting in concatemeric FNA species with dameters of several microns. Such species can be entrapped within macroporous sol-gel derived materials with micron-sized pores with minimal leaching, thus allowing for detection of a wide range of molecules, including biomolecules. Optimal materials for entrapment of FNA superstructures was achieved using a high-throughput material screening method, which minimized biomolecule leaching while maintaining FNA activity. Using an optimized material, concatemeric aptamer superstructures were entrapped within macroporous monolithic columns for flow-based detection of small molecules and proteins, extending the range of analytes that can be analyzed using biohybrid monolithic columns. Preliminary studies on the formation and properties of a DNAzyme superstructure for detection of E. coli detection were also performed, which provided valuable information on factors that must be controlled to allow reproducible fluorescence-based detection of E. coli using the crude intracellular matrix as the target. / Thesis / Master of Science (MSc)

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography. / AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

<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.</p><p>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 <i>Toxoplasma gondii</i> surface antigen through hydrophobic interaction, lipids were added either <i>in vivo</i> via <i>E. coli</i> expression, or <i>in vitro</i> 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<i> Neospora caninum</i> 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.</p><p>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 <i>in vitro</i> 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-Z_HER2/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-(Z_HER2/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 <i>in vivo</i>, 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 <i>in vivo</i> imaging and radiotherapy.</p>

Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to 'negatively' de-activate or 'positively' activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450's, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.

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.

Está estabelecido que o processo de glicosilação é essencial para a conformação estrutural e função efetora dos anticorpos. Entretanto, não está completamente claro como diferenças nos carboidratos ligados aos anticorpos podem interferir na sua atividade biológica. Foi previamente descrito que anticorpos IgG1 murinos podem ser divididos em anafiláticos ou não-anafiláticos, de acordo com a sua capacidade de induzir in vivo reação de anafilaxia. Somado a isso, foi verificado que a cadeia de oligossacarídeos N-ligada à molécula de IgG1 é fundamental para a manutenção da sua função efetora. O objetivo do presente trabalho é estudar diferenças estruturais entre os subtipos de IgG murinos que poderiam determinar a sua atividade biológica. O seqüenciamento dos nucleotídeos que codificam os domínios CH2 e CH3 dos dois subtipos de IgG1 permitiu constatar homologia de 100% dessas regiões nas duas moléculas estudadas. Entretanto, ao analisar o padrão de carboidratos N-ligados aos anticorpos IgG1 foi observado maior conteúdo de ácido siálico e fucose na cadeia N-ligada ao anticorpo anafilático em relação à do não-anafilático. Contudo, a remoção de resíduos de ácido siálico por tratamento enzimático do anticorpo IgG1 anafilático resultou na perda da capacidade desta molécula de induzir desgranulação celular in vitro e reação anafilática in vivo, semelhante ao anticorpo IgG1 deglicosilado. Em contraste, a remoção de fucose não afetou a sua função anafilática. A análise por PCR em tempo real da expressão dos genes das enzimas envolvidas no processo de glicosilação das proteínas revelou menor expressão gênica de algumas glicosidases, principalmente as sialiltransferases, no hibridoma e linfócitos B secretores do subtipo IgG1 não-anafilático em relação ao obtido no hibridoma e linfócitos B que secretam a IgG1 anafilática. Além disto, foi observada menor atividade enzimática das sialiltransferases obtidas do hibridoma produtor da IgG1 não-anafilática em relação à do hibridoma que produz a IgG1 anafilática. Em conjunto, estes resultados comprovam que a capacidade de anticorpos IgG1 murinos de induzir anafilaxia é diretamente dependente do conteúdo de ácido siálico presente na cadeia de oligossacarídeos ligada à região Fc do anticorpo, além disso sugerem fortemente que essa maior sialilação observada no tipo anafilático seja resultante da maior expressão gênica destas enzimas e assim da sua atividade enzimática no momento da síntese dos anticorpos. / It is well established that the glycosylation process is essential for the structural conformation and effector function of the antibodies. However, it is quite clear how differences in the carbohydrates attached to the antibodies may interfere with their biological activities. It was previously reported that murine IgG1 antibodies can be divided into anaphylactic or nonanaphylactic according to their ability to induce anaphylaxis. Furthermore, it was demonstrated that the oligosaccharide chain N-linked to the IgG1 is essential for its conformation and biological activity. The objective of this work is to study structural differences between these subtypes of murine IgG1 that could determine their biological activity. The sequencing of the nucleotides encoding the CH2 and CH3 domains of these two subtypes of IgG1 showed 100% of homology in the Fc regions of these molecules. In contrast, the analysis of the carbohydrates N-linked to the IgG1 antibodies demonstrated higher sialic acid and fucose contents in the chain attached to the anaphylactic antibody than in the nonanaphylactic IgG1. However, the removal of sialic acid residues by enzymatic treatment of anaphylactic IgG1 antibody resulted in the abrogation of its ability to induce mast cells degranulation in vitro and anaphylactic reaction in vivo as observed to deglycosylated IgG1 antibody. On the other hand, the removal of fucose did not change the anaphylactic activity. The analysis by real time PCR of the gene expression of enzymes that are involved in the protein glycosylation showed lower gene expression of some glycosyltransferases, mainly sialyltransferases, in the hybridoma and B lymphocytes that produce the non-anaphylactic IgG1 compared to those verified in the hybridoma and B cells producer of the anaphylactic IgG1. Furthermore, it was verified lower enzymatic activity of sialyltransferases purified from the hybridoma producer of the non-anaphylactic IgG1 in relation to the hybridoma producer of the anaphylactic antibody. Together, these results prove that the ability of murine IgG1 to induce anaphylaxis is directly dependent of the sialic acid content in the carbohydrate core attached to the antibody Fc region. It is also strongly suggested that this higher sialylation observed in the anaphylactic IgG1 may be resultant of the higher gene expression and enzymatic activity of some sialyltransferases during the antibody synthesis.