The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

<p>With the full sequence of the human genome now available, anexciting era in biomedical research has started. The sequenceprovides information about all our genes and greatly increasesthe scope to compare genetic activities in different cells, toanalyze genetic variation between individuals and betweendifferent species and, most importantly, to investigatesystematically the whole genome in a gene-by-gene manner, andthus increase our understanding of gene function.</p><p>This thesis describes studies in which developments weremade in several areas of functional genomics. Messenger RNAlevels were analyzed by the use of an amplification procedure,in which the 3´-ends of the transcripts were selected inorder to amplify the mRNA population in an unbiased fashion. Bysonicating cDNA originating from expressed mRNA, uniformlysized representatives of the transcripts,“signaturetags”, were obtained. The mRNA levels in the original mRNApopulation correlated well with the levels in the amplifiedmaterial, as verified by microarray analysis and realtimequantitative PCR. The expressed transcripts can be identifiedusing pyrosequencing, by comparing the obtained sequenceinformation from the signature tags to information contained invarious sequence databases. In one of the articles, the use ofpyrosequencing is illustrated by efforts to find genes involvedin the disease progression of atherosclerosis.</p><p>More challenging than the study of mRNA levels is to analyzewhen, where and how proteins fulfill their wide-ranging rolesin all the various cellular processes. Proteins are morecomplex biomolecules than mRNA, each having unique properties.Current techniques for studying proteins need much improvement,and are often limited to investigations of a specific portionof the proteome. One approach for studying the whole proteomeis to systematically generate reagents with specific affinityfor the proteins encoded by the genome, one by one. Theaffinity reagents can be used as flags for their targets,providing a flag-specific detection system, so that the targetproteins can be sub-cellularly localized in the majority ofhuman tissues in an array format. One of the articles includedin the thesis presents a pilot project for large-scale affinityreagent production. The aim was to provide a sound basis forwhole proteome studies, but as a pilot study this investigationwas limited to the proteins encoded by human chromosome 21. Allputative genes on the chromosome were subjected to antibodygeneration in a systematic manner. Small, uniform, and easilyproduced representative portions of the full-length proteinswere expressed. These were denoted“Protein EpitopeSignature Tags”and were designed to be unique for theirfull-length counterparts. The antibodies were produced inrabbits and two of the articles in the thesis discuss differentapproaches for affinity purification of the antibodies toachieve the highest possible specificity towards the targets.The resulting“mono-specific”, but still“multi-epitope”, antibodies can be used for a widerange of additional biochemical studies, such as protein arrayand protein pull-out analyses.</p><p><b>Keywords:</b>functional genomics, 3´-end signaturetags, pyrosequencing, amplification, PrEST, chromosome 21,polyclonal antibodies, dual expression, affinitypurification.</p>

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4-15% gradient polyacrylamide gels. Both DNase and RNase were dependent on metal co-factors for activity. The metal ion Mg2+ was the preferred ion for the DNase, whereas for the RNase, Zn2+ and Mn2+ yielded an increase in enzyme activity over that with Mg2+. The purified DNase demonstrated maximum activity at pH 6.0 with no activity at pH 2.0 or 10.0. The RNase exhibited two peaks of maximum activity, on at pH 3.0 and the other at pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65oC. The optimal temperature for the RNase was 70oC. The molecular of the DNase and RNase were determined to be 56 kDa and 69kDa respectively using a Sephadex G-75 column. A standard curve was generated using several standard proteins of known molecular weight.

Diese Arbeit hatte es zum Ziel, eine neue und effiziente Affinitätsplattform für die Aufreinigung und Isolierung von Proteinen basierend auf nicht‐porösen und stabilen Korund‐Partikeln zu entwickeln. Das Rohmaterial wurde kovalent mit Proteinbindern modifiziert, um so die Isolierung spezieller Target‐Proteine aus komplexen biologischen Proben zu realisieren. Für die erste Modifizierungsschicht auf der Korundoberfläche wurden verschiedene Phosphonsäuren und Silane untersucht. Anschließend wurde der etablierte bifunktionale Crosslinker Glutaraldehyd mit dem biokompatibleren verzweigten Polyglycerol (PG) verglichen. Es konnte gezeigt werden, dass Polyglycerol eine hervorragende Alternative zu Glutaraldehyd darstellen kann. Für die angestrebte Anwendung als neues Tool für die Antikörperreinigung wurde humanes IgG mit Protein‐A‐funktionalisierten Korundpartikeln erfolgreich aus Humanplasma isoliert. Um das Anwendungsspektrum der neuen Korundmethode auszuweiten, wurde im zweiten Teil dieser Arbeit eine Affinitätsplattform für die Isolierung Polyhistidin‐getaggten Proteinen basierend auf Metallionen‐funktionalisiertem Korund entwickelt und angewendet. Hauptkriterien dieser Methodenentwicklung waren die schnelle, effiziente und ökonomische Aufreinigung rekombinanter Proteine aus bakteriellen Lysaten in einem säulenfreien Format. Als Modellsystem wurde Polyhistidin‐getaggtes Protein A/G (His6‐PAG) mit Rinderserumalbumin versetzt, was die Entwicklung eines optimierten Protokolls für die Isolierung rekombinanter Proteine ermöglichte. Im direkten Vergleich mit kommerziellen Ni‐NTA‐Agarose‐Beads zeigten die Korundpartikel höhere Proteinreinheiten. Abschließend konnte gezeigt werden, dass Zink eine ideale Alternative als Metallion darstellt, um etwaige Nachteile des Nickels in der Anwendung von Life‐Science‐Methoden zu umgehen und eine zukunftsträchtige Methode mit weiterem Potenzial zu realisieren. / This work aimed to develop and establish a novel efficient affinity platform for protein purification based on nonporous, stable, and easily available corundum powder. The material was functionalized covalently with protein binders to isolate and enrich specific proteins from complex biological matrices. Phosphonic acids and silanes were tested as the first modification layer. In the next step, the well‐known bifunctional crosslinker glutaraldehyde was compared with a more biocompatible, hyperbranched polyglycerol (PG). It could have been shown that oxidized polyglycerol is an excellent alternative to glutaraldehyde. Human IgG was purified with protein A functionalized corundum from crude human plasma for the initial purpose of antibody isolation. To broaden the application of functionalized corundum, a novel purification platform for the isolation of poly His‐tagged proteins based on metal‐ion‐functionalized corundum was established.The main goal of this approach was the efficient, economical, and fast purification of recombinant proteins in a column‐free format that can also easily be performed in moderately equipped laboratories. As a model system for method optimization, His‐tagged protein A/G (His6‐PAG), mixed with bovine serum albumin (BSA), was established leading to a purification protocol with minimal nonspecific binding by the variation of the imidazole content in the used binding and washing buffers. Corundum in direct comparison with standard Ni‐NTA agarose beads generated higher purities of isolated proteins. Finally, it was shown that zinc‐functionalized corundum is another efficient approach to circumvent potential negative effects of nickel use in life science applications.

Les fichiers qui accompagnent mon document sont des tableaux supplémentaires réalisés avec Excel (Microsoft Office), dans la version papier du mémoire ces fichiers sont sur un CD-ROM. / Une large gamme d’événements cellulaires est régulée par la SUMOylation des protéines. Cette modification post-traductionnelle est impliquée dans le cancer notamment dans la leucémie promyélocytaire aigue. À ce jour, peu d’études à grande échelle ont porté sur l’identification des sites de modification. Ce mémoire présente une approche protéomique quantitative unique qui combine une double purification par affinité au niveau des protéines cibles ainsi que des peptides modifiés. L’approche la plus répandue de purification des protéines SUMOylés implique l’utilisation d’une forme de SUMO modifié avec une étiquette (His6-SUMO). A ce jour, les approches permettant l’enrichissement au niveau peptidique nécessite une forme mutante de SUMO. Notre analyse consiste à premièrement enrichir en protéines SUMOylés dans les cellules humaines vierges ou sur exprimant His6-SUMO-1/3 en présence ou pas de trioxyde de diarsenic, un traitement de leucémie promyélocytaire aigue. Par la suite, les échantillons sont digérés et les peptides obtenus des protéines SUMOylés conservent un branchement caractéristique. Les peptides sont soit immunoprécipités avec un anticorps spécifique au branchement SUMO ou directement analysés par nano LC/LC-MS/MS par un spectromètre de masse LTQ-Orbitrap. Une analyse manuelle des données révèle des fragments caractéristiques correspondant à la chaîne latérale de SUMO. L’originalité de l’approche réside dans l’identification quantitative et sans ambigüité des sites de SUMOylation. Cette approche a permis l’identification de 17 et 3 sites de SUMO-3 et SUMO-1 respectivement dans les cellules HEK293. Finalement, la SUMOylation de PML est induite suite au traitement d’arsenic. / A wide range of cellular events are regulated by protein SUMOylation. This posttranslational modification was involved in APL (acute promyelocytic leukemia). Only a few large scale studies in mammalian cells have focused on identifying the conjugation sites. This thesis presents a unique quantitative proteomics approach that combines double affinity purification at the protein and peptide level. A common approach to purification of SUMOylated proteins involves the use of a tagged SUMO (His6-SUMO). To date, the SUMO peptide isolation is addressed using an engineered SUMO. In presence or absence of arsenic trioxide, a treatment of APL, mock and His6-SUMO1/3 expressing cells are lysed and the SUMOylated proteins are isolated under denaturing conditions. Subsequently, these samples are digested and the peptides bearing the modification site bear a specific SUMO stub. They are either immunoprecipitated with an anti SUMO stub antibody or directly analyzed by nano LC coupled to an LTQ-Orbitrap mass spectrometer. Manual analysis of the data reveals characteristic fragmentation corresponding to the side chain of SUMO. The originality of the approach lies in the quantitative and unambiguous identification of SUMOylation sites in vivo. This approach allowed the identification of 17 and 3 sites of SUMO-3 and SUMO-1, respectively, in HEK293 cells. Finally, PML was identified as the major SUMOylation target following arsenic treatment.

Die Epithelien vielzelliger Organismen bilden eine funktionelle Grenzschicht, die für die Homöostase innerhalb und für den spezifischen Stoffaustausch zwischen den Kompartimenten verantwortlich ist. Der interzelluläre Spalt zwischen Epithelzellen wird durch tight junctions verschlossen, die eine selektive Permeabilitätsbarriere bilden. Da viele Krankheiten auf eine Dysfunktion der Barriere zurückzuführen sind, ist eine genaue Kenntnis der molekularen Zusammensetzung der tight junctions aus pharmakologischer Sicht von großem Interesse. In dieser Arbeit wurden Anreicherungsstrategien entwickelt, die eine Proteomanalyse der tight junction-Proteine erlauben. Der Fokus wurde dabei auf die Claudine und Tricellulin gelegt, die als transmembranale Proteine das molekulare Rückgrat der tight junctions bilden. Durch Affinitätsreinigung gelang erstmals eine Anreicherung verschiedener Claudine, die durch Massenspektrometrie identifiziert wurden. Die metabolische Markierung der Proteine mit stabilen Isotopen (SILAC) erlaubte die quantitative Diskriminierung von Proteinen, die unspezifisch an das Matrixmaterial banden. Von den potentiellen Interaktionspartnern der Claudine wurden Integrin-a3, SUMO-1 und Sphingosinkinase 2 ausgewählt, um deren Interaktion mit Claudinen weiter zu verifizieren. Es wurden keine Hinweise auf Wechselwirkungen zwischen Claudinen und Integrin-a3 sowie SUMO-1 gefunden, während die Interaktion von Claudinen mit Sphingosinkinase 2 weder bestätigt noch ausgeschlossen werden konnte. Ferner wurde eine Affinitätsreinigung durchgeführt, um Interaktionspartner von Tricellulin anzureichern. Durch die quantitative massenspektrometrische Analyse wurde ausschließlich Claudin-4 nicht aber Claudin-3 und 7 als potentieller, spezifischer Interaktionspartner von Tricellulin identifiziert. Es wurde aber gezeigt, dass die Kombination aus Affinitätsreinigung und quantitativer Massenspektrometrie einen wertvollen Beitrag zur Entschlüsselung von Protein-Komplexen leisten kann. / Epithelia function as specialized barriers that separate different compartments within multicellular organisms and regulate the specific exchange of substances between them. The intercellular space between adjacent epithelial cells is sealed by tight junctions forming a permeability barrier. Dysregulation of the barrier occurs in a variety of diseases. Hence, a deeper knowledge is required of the molecular composition of tight junctions, in particular with respect to pharmacological applications. In the present study, new enrichment strategies have been established that allow the proteomic analysis of tight junction proteins. Special emphasis was placed on claudins and tricellulin as these transmembrane proteins constitute the molecular backbone of the tight junctions. For the first time, using an affinity purification, the enrichment of several claudins was accomplished that were identified by mass spectrometry. The metabolic labeling of proteins with stable isotopes (SILAC) allowed the quantitative discrimination of proteins that bound unspecifically to the matrix. Integrin-a3, SUMO 1 and sphingosin kinase 2 were chosen for further verifications from the proteins considered to potentially interact with claudins. While there was no evidence for an association of claudins with integrin-a3 and SUMO-1, an interaction of claudins with sphingosin kinase 2 could be neither confirmed nor disproved. Furthermore, an affinity purification was performed in order to enrich interaction partners of tricellulin. Claudin-4 was identified as a specific, potential interaction partner of tricellulin by quantitative mass spectrometric analysis whereas claudin 3 and -7 were determined to be enriched unspecifically. The present study demonstrates that a combination of affinity purification and quantitative mass spectrometry can substantially contribute to the elucidation of protein complexes.

HTML clipboard The great advances in DNA technology, e.g. sequencing and recombinant DNA techniques, have given us the genetic information and the tools needed to effectively produce recombinant proteins. Recombinant proteins are valuable means in biotechnological applications and are also emerging as alternatives in therapeutic applications. Traditionally, monoclonal antibodies have been the natural choice for biotechnological and therapeutic applications due to their ability to bind a huge range of different molecules and their natural good affinity. However, the large size of antibodies (150 kDa) limits tissue penetration and the recombinant expression is complicated. Therefore, alternative binders with smaller sizes have been derived from antibodies and alternative scaffolds. In this thesis, two structurally similar domains, Zbasic and ABDz1, have been used as purification tags in different contexts. They are both three-helical bundles and derived from bacterial surface domains, but share no sequence homology. Furthermore, by redesign of the scaffold used for ABDz1, a molecule intended for drug targeting with extended in-vivo half-life has been engineered. In Papers I and II, the poly-cationic tag Zbasic is explored and evaluated. Paper I describes the successful investigation of Zbasic as a purification handle under denaturating conditions. Moreover, Zbasic is evaluated as an interaction domain in matrixassisted refolding. Two different proteins were successfully refolded using the same setup without individual optimization. In Paper II, Zbasic is further explored as a purification handle under non-native conditions in a multi-parallel setup. In total, 22 proteins with varying characteristics are successfully purified using a multi-parallel protein purification protocol and a robotic system. Without modifications, the system can purify up to 60 proteins without manual handling. Paper I and II clearly demonstrate that Zbasic can be used as an interaction domain in matrix-assisted refolding and that it offers a good alternative to the commonly used His6-tag under denaturating conditions. In paper III, the small bifunctional ABDz1 is selected from a phage display library. Endowed with two different binding interfaces, ABDz1 is capable of binding both the HSA-sepharose and the protein A-derived MabSelect SuRe-matrix. The bifunctionality of the domain is exploited in an orthogonal affinity setup. Three target proteins are successfully purified using the HSA-matrix and the MabSelect SuRe-matrix. Furthermore, the purity of the target proteins is effectively improved by combining the two chromatographic steps. Thus, paper III shows that the small ABDz1 can be used as an effective purification handle and dual affinity tag without target specific optimization. Paper IV describes the selection and affinity maturation of small bispecific drug-targeting molecules. First generation binders against tumor necrosis factor-α are selected using phage display. Thereafter on-cell surface display and flow cytometry is used to select second-generation binders. The binding to tumor necrosis factor-α is improved up to 30 times as compared to the best first generation binder, and a 6-fold improvement of the binding strength was possible with retained HSA affinity. Paper III and IV clearly demonstrate that dual interaction surfaces can successfully be grafted on a small proteinaceous domain, and that the strategy in paper IV can be used for dual selection of bifunctional binders. / <p>QC20100610</p>

Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated. Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived fromStaphylococcus aureusprotein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of thePlasmodium falciparumblood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies forin vivoorin vitrolipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived fromToxoplasma gondii. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation. Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product. Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy. We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against theP. falciparumPf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein. <b>Keywords</b>: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,Staphylococcus aureusprotein A, Subunit vaccine,Toxoplasma gondii