The present work demonstrates how a peptide-based material can be obtained for the biosorptive recovery of metals from contaminated industrial wastewater. Starting with Phage surface display for the initial identification and optimization of gallium-binding peptides, all the following application-focussed experiments are based on chemically synthesized peptides. Two chromatography-based biopanning methods for the identification of gallium-binding peptides from a commercial phage display library were developed. Five gallium-binding peptide sequences were identified and evaluated to show good gallium-binding properties. Furthermore, the biosorption of free gallium and arsenic by gallium-binding bacteriophage clones was investigated. A large influence of the pH-value on the respective interactions was demonstrated. Mutagenesis experiments were also carried out for a bacteriophage clone expressed peptide, in which a cysteine pair systematically replaced amino acids. Biosorption experiments with the resulting seven different bacteriophage mutants suggested a relationship between the rigidity of the peptide structure and the gallium-binding properties. In isothermal titration experiments, the thermodynamics of the interaction between gallium and the peptides as chemically synthesized derivatives were characterized, independent of the bacteriophage. The peptides differed strongly in their interaction with gallium, and in some cases, the complex formation with gallium depended strongly on the surrounding buffer conditions. The peptide with the amino acid sequence NYLPHQSSSPSR has particularly promising gallium-binding properties. Computer modeling suggests the probable structure of the peptide in aqueous solution and postulates a possible binding site for gallium. The side-selective and covalent immobilization of the peptides on a polystyrene matrix led to the creation of a biocomposite for the biosorptive recovery of gallium. The sorption performance and desorbability of the peptide-based biosorption materials were determined in studies with model solutions and real waters from the semiconductor industry.  :EIDESSTATTLICHE VERSICHERUNG II SUMMARY 7 CHAPTER I. 8 Utility of biotechnological approaches in resource technology 9 Phage Surface Display for the recovery of inorganic binding peptides 14 Gallium – Example of a high-tech metal 22 Aims and context of the present work 22 CHAPTER II. 25 Author contributions 25 Abstract 25 Introduction 26 Materials and Methods 28 2.1 Phage Display Library system 28 2.2 Biopanning experiments 29 2.3 Single clone identification 31 2.4 Single clone binding studies 31 Results 32 3.1 Immobilization of gallium ions 32 3.2 Biopanning experiments 33 3.3 Single clone binding studies 38 Discussion 39 4.1 Gallium ions as biopanning target 39 4.2 Phage clone selection 40 Conclusion 44 Acknowledgements 45 CHAPTER III. 46 Author contributions 46 Abstract 47 Introduction 47 Materials and Methods 49 2.1 Handling of phage display library clones 49 2.2 Site-directed mutagenesis experiments 50 2.3 Biosorption experiments 51 Results and Discussion 52 3.1 Experimental context 52 3.2 Original phage clone characterization 53 3.3 Site-directed mutagenesis experiments 56 3.4 Mutant phage clone characterization 57 Conclusions 59 Acknowledgements 60 CHAPTER IV. 61 Author contributions 61 Textual and graphical abstract 62 Introduction 63 Methods 65 2.1 Peptides 65 2.2 Isothermal titration microcalorimetry (ITC) 65 2.3 Preparation of peptide conjugates 65 2.4 Biosorption studies 66 2.5 Model calculation of peptide C3.8 67 Results and Discussion 68 3.1 Interaction studies of free peptides in solution 68 3.2 Biosorption studies with peptide polystyrene conjugates 71 3.2.1 Covalent and site-selective immobilization of peptides 71 3.2.2 Interaction of peptide conjugates with gallium 72 3.2.3 Interaction of peptide conjugates with arsenic 73 3.2.4 Continuous experiments 73 3.3 Model calculation for peptide C3.15 75 Counclusion 76 Acknowledgment 77 CHAPTER V. 78 Obtained insights for the selection of metal-binding peptides in biopanning experiments 79 Conclusions for the development of peptide-based materials for the biosorptive recovery of metal ions from aqueous solutions 81 REFERENCES 85 APPENDIX 94 SUPPORTING INFORMATION FOR CHAPTER IV 94 LIST OF FIGURES 99 LIST OF TABLES 100 LIST OF ABBREVIATIONS 101 LIST OF CHEMICALS 104 ACKNOWLEDGEMENTS 106 CURRICULUM VITAE 109 LIST OF PUBLICATIONS 111

Development of nanochemical tools targeting receptors in the tumor microenvironment Targeting the receptors in the tumor microenvironment is crucial for the future development of targeted therapies, precision medicine and immunotherapy of cancer. The options available now are, however, limited by the availability of specific ligands. The advances in the field strongly rely on the use of antibodies and genetic modifications of immune cells. Availability of small molecules targeting the receptors of interest would allow further development of alternative strategies as well as deepen our understanding of the underlying mechanisms of cancer development, progression and clearance. In the search for new small-molecule ligands and their use for receptor targeting, the prostate-specific membrane antigen (PSMA) and the immune receptors CD3 and CD64 were selected as model targets. The selected method - the phage display of bicyclic peptides - utilizes chemical modification of the displayed three-cysteine peptides to achieve their cyclization and formation of bicycles. The panning of a peptide library displayed on the phages and probed with PSMA revealed a reproducibly-selected amino acid sequence. Interestingly, the phage clone carrying this sequence was a specific binder of PSMA, but the synthesized peptide alone...

No description available.

Les communautés bactériennes sont constituées d’un grand éventail d’espèces pouvant interagir entre elles dans des environnements spatialement hétérogènes tels que le sol, les plantes ou l'intestin humain. À quel point ces interactions stimulent ou entravent la diversité du microbiome demeure inconnu. Historiquement, deux hypothèses ont été proposées pour expliquer comment les interactions interespèces pourraient influencer la diversité. L’hypothèse ‘l’écologie contrôle’ (EC) prédit une relation négative, dans laquelle l'évolution ou la migration de nouvelles espèces est freinée à mesure que les niches se saturent. En revanche, l’hypothèse ‘la diversité engendre la diversité’ (DBD) prédit une relation positive, où la diversité existante favorise l'accumulation d'une plus grande diversité à travers des interactions telles que la construction de niche. De nombreuses études ont investigué ces modèles chez les vertébrés ou les plantes, et certaines les ont testés sur des bactéries en culture ; mais le modèle qui régit les communautés bactériennes naturelles demeure inconnu. En utilisant les données du gène ARN ribosomique 16S provenant d’un large éventail de microbiomes, j'ai montré une relation positive générale entre la diversité des taxons et la diversité des communautés de niveaux taxonomiques plus élevés. Cette observation est conforme à l’hypothèse du DBD, mais cette tendance positive plafonne à des niveaux élevés de diversité en raison des limites physiques de la niche. Ensuite, j'ai observé que le modèle DBD restait valide à une résolution plus fine, en analysant la variation génétique intra espèce dans les métagénomes des microbiomes intestinaux humains. Conformément au DBD, j'ai observé que le polymorphisme génétique ainsi que le nombre de souches intra espèces étaient positivement corrélés avec la diversité Shannon de la communauté. Dans le chapitre 3, j'ai examiné les interactions antagonistes entre V. cholerae et ses phages virulents et la manière dont ces interactions affectaient le cours de l’infection et la diversité génétique de V. cholerae chez les patients infectés. J'ai quantifié les abondances relatives de V. cholerae et des phages virulents associés dans plus de 300 métagénomes provenant de selles de patients atteints de choléra, tout en tenant compte de leur exposition aux antibiotiques. Les phages et les antibiotiques ont supprimé V. cholerae et ont été associés à une déshydratation légère chez les patients. J'ai également investigué les mécanismes de défense contre les phages dans V. cholerae et découvert que les éléments connus de résistance aux phages (integrative conjugative elements, ICEs) étaient associés à de faibles rapports phage: V. cholerae. J’ai pu montrer aussi que lorsque les ICEs ne sont pas détectés, la résistance aux phages semble être acquise par l’accumulation de mutations ponctuelles non synonymes. Mes résultats valident que les phages virulents sont un facteur qui protège contre le choléra tout en sélectionnant la résistance dans le génome de V. cholerae. / Bacterial communities harbor a broad range of species interacting within spatially heterogeneous environments such as soil, plants or the human gut. The extent to which these interactions drive or impede microbiome diversity is not well understood. Historically, two hypotheses have been suggested to explain how species interactions could influence diversity. The ‘Ecological Controls’ (EC) hypothesis predicts a negative relationship, where the evolution or migration of novel species is constrained as niches become filled. In contrast, the ‘Diversity Begets Diversity’ (DBD) hypothesis predicts a positive relationship, with existing diversity promoting the accumulation of further diversity via niche construction and other interactions. Many studies investigated these models in vertebrates or plants, some focused on cultured bacteria, but we still lack insights into how natural communities are assembled in the context of these two hypotheses. Using 16S RNA gene amplicon data across a broad range of microbiomes, I showed a general positive relationship between taxa diversity and community diversity at higher taxonomic levels, consistent with DBD. Due to niche’ limits, this positive trend plateaus at high levels of community diversity. Then, I found that DBD holds at a finer resolution by analyzing intra-species strain and nucleotide variation in sampled metagenomes from human gut microbiomes. Consistent with DBD, I observed that both intra-species polymorphism and strain number were positively correlated with community Shannon diversity. In Chapter 3, I investigated the antagonistic interactions between V. cholerae and its virulent phages and how these interactions affect the course of the infection and the within V. cholerae genetic diversity in natural infections. I quantified relative abundances of Vibrio cholerae (Vc) and associated phages in 300 metagenomes from cholera patients stool, while accounting for antibiotic exposure. Both phages and antibiotics suppressed V. cholerae and were inversely associated with severe dehydration. I also looked at V. cholerae phage-defense mechanisms and found that known phage-resistance elements (integrative conjugative elements, ICEs) were associated with lower phage:V. cholerae ratios. In the absence of detectable ICEs, phages selected for nonsynonymous point mutations in the V. cholerae genome. My findings validate that phages may protect against severe cholera while also selecting for resistance in the V. cholerae genome within infected patients.

No description available.

Diese Arbeit beschreibt die Kombination von Phagen-Display-Biopanning und Illumina Next-Generation DNA-Sequencing (NGS) zur Identifizierung peptidbasierter Adhäsionsdomänen für Polypropylen (PP). Eine Biopanning-Runde gefolgt von NGS liefert PP-bindende Peptide, die durch Sanger-Sequenzierung nicht erkennbar sind. NGS bietet den Vorteil eines enorm umfangreichen Datensatzes, welcher tiefgreifende Sequenzanalysen erlaubt. Die selektierten Sequenzen werden als wasserbasierte Primer für PP–Metallhaftung zur Vorbehandlung von PP-Oberflächen eingesetzt und erhöhen die Haftfestigkeit um 100 % gegenüber nicht vorbehandeltem PP. / This thesis describes the combination of phage display biopanning and Illumina Next-Generation DNA-Sequencing (NGS) to identify peptide-based adhesion domains for polypropylene (PP). One round of biopanning followed by NGS yields PP-binding peptides that are undetectable by Sanger sequencing. NGS has the advantage of an extensive data set, which allows in-depth sequence analysis. The selected peptide sequences are then used as water-based primers for PP metal adhesion for the pretreatment of PP surfaces and increase the adhesion by 100% compared to non-pretreated PP.

Im Rahmen der vorliegenden Arbeit wird eine neue, kostengünstige und effiziente Strategie zur selektiven Beschichtung gedruckter Muster auf Papier entwickelt. Mittels Phagen-Display werden Peptide identifiziert, die materialspezifische Adsorption zeigen und effektiv zwischen Cellulose als Hauptbestandteil von Papier und dem Toner eines mit einem handelsüblichen Laserdrucker gedruckten Musters unterscheiden können. Diese genetisch selektierten 12mer Peptide können selektiv entweder nicht bedruckte Cellulose oder gedruckte Tonerstrukturen beschichten. Es werden vielfältige Adsorptionsuntersuchungen einzelner Peptide an den jeweiligen separaten Oberflächen und an gedruckten Mustern durchgeführt und diskutiert. Des Weiteren wird eine Ligationschemie mit Triazolindion genutzt, um z. Bsp. Farbstoffe oder funktionale Peptide selektiv auf einer Oberflächenbeschichtung zu lokalisieren. Diese Methodik bietet einen einfachen Zugang zur Funktionalisierung von Mustern auf papierbasierten Materialien, wodurch neue Wege zur Realisierung kostengünstiger diagnostischer oder biomedizinischer Geräte aufgezeigt werden können. / Within the scope of this thesis, a new, cost-effective and efficient strategy for the selective coating of printed patterns on paper is developed. Phage display biopanning identifies peptides that show material selective adsorption, effectively distinguishing between cellulose of paper and printed toner of standard office laser printers. These genetically selected 12mer peptides can selectively coat either non-printed cellulose or printed toner patterns. Numerous adsorption studies of individual peptides on the respective separated surfaces and on printed patterns are carried out and discussed. Furthermore, triazolindione ligation chemistry is exploited to introduce e.g. dyes or functional peptides selectively to the coatings. The strategy offers an easy access toward patterned functionalization of paper based materials, which potentially is of relevance for low cost diagnostics or biomedical devices.

A prevalent morphology in the microscopic world of artificial microswimmers, bacteria and viruses is that of a helix. The intriguingly different physics at play at the small scale level make it necessary for bacteria to employ swimming strategies different from our everyday experience, such as the rotation of a helical filament. Bio-inspired microswimmers that mimic bacterial locomotion achieve propulsion at the microscale level using magnetically actuated, rotating helical filaments. A promising application of these artificial microswimmers is in non-invasive medicine, for drug delivery to tumours or microsurgery. Two crucial features need to be addressed in the design of microswimmers. First, the ability to selectively control large ensembles and second, the adaptivity to move through complex conduit geometries, such as the constrictions and curves of the tortuous tumour microvasculature. In this dissertation, a mechanics-based selective control mechanism for magnetic microswimmers is proposed, and a model and simulation of an elastic helix passing through a constricted microchannel are developed. Thereafter, a theoretical framework is developed for the propulsion by stiff elastic filaments in viscous fluids. In order to address this fluid-structure problem, a pertubative, asymptotic, elastohydrodynamic approach is used to characterise the deformation that arises from and in turn affects the motion. This framework is applied to the helical filaments of bacteria and magnetically actuated microswimmers. The dissertation then turns to the sub-bacterial scale of bacteriophage viruses, 'phages' for short, that infect bacteria by ejecting their genetic material and replicating inside their host. The valuable insight that phages can offer in our fight against pathogenic bacteria and the possibility of phage therapy as an alternative to antibiotics, are of paramount importance to tackle antibiotics resistance. In contrast to typical phages, flagellotropic phages first attach to bacterial flagella, and have the striking ability to reach the cell body for infection, despite their lack of independent motion. The last part of the dissertation develops the first theoretical model for the nut-and-bolt mechanism (proposed by Berg and Anderson in 1973). A nut being rotated will move along a bolt. Similarly, a phage wraps itself around a flagellum possessing helical grooves, and exploits the rotation of the flagellum in order to passively travel along and towards the cell body, according to this mechanism. The predictions from the model agree with experimental observations with respect to directionality, speed and the requirements for succesful translocation.

Fagmider är ett kraftfullt verktyg som använts för att uttrycka heterologa proteiner på fagvirus i metoder som exempelvis riktad evolution via fagdisplay av proteinbinliotek. Monovalent uttryck genom fagemider lider av ett överflöd av kala fager som är svåra att rensa bort. Effektiviteten hos detta system kan förbättras genom att använda tekniker för att selektivt fånga uttryckande fag. Biotinylering och infångning med streptavidin kan vara ett användbart verktyg för sådana ändamål och enzymatisk biotinylering med AviTag™-BirA-systemet är ett lovande alternativ. En fagemidvektor skapades genom amplifiering och kloning av sekvensen av AviTag™ in i en linjäriserad pAffi1#336-vektor. Sekvenser av två affikroppar, Zher2 och Zwt, som binder till två olika mål, Her2 och trastuzumab, klonades in i denna Avi-märkta vektor. Denna sammansatta fagemidvektor transformerades därefter till tre Escherichia coli-stammar: XL1Blue, ER2738, TG1. En plasmid innehållande BirA-enzym avsett för platsspecifik biotinylering av proteinerna som bär AviTag™ samtransformerades tillsammans med fagemiden i vissa transformationer för att undersöka biotinylering in vivo. Produktionsnivåerna för var och en av dessa stammar tillsammans med deras tillväxtkurvor beräknades för att analysera om plasmidbelastningen påverkar någon av stammarnas tillväxt. Det observerades att XL1Blue kunde växa i en takt som var jämförbar med de andra stammarna och samtidigt lyckades producera tillräckligt högre titrar. Superinfektion av hjälparfager för dessa produktioner var också mycket lovande. ER2738 och TG1 var avsevärt dåliga, den förra i termer av alla aspekter och den senare saknade bra superinfektionskarraktär men uppvisade anmärkningsvärt högre tillväxt och avsevärt högre titrar. Fagen biotinylerades in vitro genom att använda BirA som producerades och renades internt och dess biotinyleringsgrad jämfördes med den biotinylerade in vivo fagen som producerades i dessa tre stammar. Dessa fager utvärderades för deras förmåga att binda till sina mål och deras grad av biotinylering. Ett märkbart mönster som observerades fag visade minskad bindning till humant serumalbumin, vilket gjorde deras målbindning svår att tolka. / Phagemids have been a convenient and powerful tool used to display heterologous proteins on phage in methods such as directed evolution by phage display of protein libraries. Monovalent display through phagemids suffers from an overabundance of bald phage which are difficult to deplete. The efficiency of this system can be improved by using techniques to selectively capture expressing phage. Biotinylation and capture using streptavidin can be a useful tool for such purposes and enzymatic biotinylation using the AviTag™-BirA system is a promising option. A phagemid vector was created by the amplification and cloning of the sequence of AviTag™ into a linearized pAffi1#336 vector. Sequences of two affibodies, Zher2 and Zwt, binding to two different targets, Her2 and human IgG, were cloned into this Avi-tagged vector. This assembled phagemid vector was subsequently transformed into three Escherichia coli strains: XL1Blue, ER2738, TG1. A plasmid containing BirA enzyme intended for site-specific biotinylation of the proteins that carry the AviTag™ was co-transformed along with the phagemid in some transformations to investigate in vivo biotinylation. The production levels of each of these strains along with their growth curves were calculated to analyse if the plasmid burden impacts either of the strains’ growth. It was observed that XL1Blue was able to grow at a pace comparable to the other strains and managed to produce sufficiently high titers. The superinfection rate of these productions was also very promising. ER2738 and TG1 were considerably poor, the former in terms of all aspects and the latter only lacking in the superinfection rate but notable displaying higher growth and considerably higher titers. The phage was biotinylated in vitro by using BirA that was produced and purified in-house and its biotinylation degree was compared against the in vivo biotinylated phage that were produced in these three strains. The phage were then evaluated for their ability to bind to their targets and their degree of biotinylation. One noticeable pattern that was observed was that the Avi- tagged phage displayed reduced binding to the Human Serum Albumin which made their target binding hard to interpret.

Under senare år har målinriktad terapi varit ett växande område inom cancerterapi som en mer målinriktad behandling än kemoterapi. Dessa behandlingar baseras främst på antikroppsbaserade läkemedel som är ganska stora och komplexa, vilket ökar den totala kostnaden för behandlingen. Därför måste man hitta en alternativ metod för både upptäckt och behandling för att hjälpa patienterna. Små affinitetsdomäner har skapats med målet att förbättra vävnadspenetrationen och samtidigt upprätthålla en hög grad av målspecificitet, vilket leder till färre biverkningar. Ett av exemplen på detta är Albumin Binding Domain-Derived Affinity Protein (ADAPT). Det har baserats på en av de albuminbindande domänerna (ABD) i streptokockproteinet G, med en storlek på 6,5 kDa. Nyligen modifierades ADAPT ytterligare för att samtidigt binda albumin och ett annat relevant målprotein av intresse, vilket tyder på en längre halveringstid i patientserum och möjliggör utveckling av nyare och terapeutiska läkemedel. I detta projekt presenteras den fjärde generationen av ADAPT-biblioteket som utformats för att ha förbättrad stabilitet. Selektioner med fagdisplay utfördes mot tre målproteiner: carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), en biomarkör för kolorektalcancer, epithelial cell adhesion molecule (EpCAM), en markör för flera gastrointestinala karcinom och trophoblast cell-surface antigen 2 (Trop2) som är överuttryckt i trippel-negativ bröstcancer. Resultaten visar bindning till CEACAM5, EpCAM och Trop2, vilket har visats med monoklonal fag-ELISA. Bindningsaffiniteten, sekundärstrukturen hos de utvalda bindarna och den bispecifika bindningen till serumalbumin återstår att utvärdera ytterligare. Projektet visar således att de ADAPTs som valts ut mot målen CEACAM5, EpCAM och Trop2 har en enorm potential för framtida kliniska tillämpningar som syftar till utveckling av diagnostik och terapi för dessa cancerbiomarkörer. / In recent years, targeted therapy has been a growing field of cancer therapy as a more targeted treatment than chemotherapy. These treatments are primarily based on antibody-based pharmaceuticals which are quite large and complex, increasing the overall cost of the treatment. Thus, an alternative method of both detection and treatment needs to be found to aid patients. Small affinity domains have been created with the goal of enhancing tissue penetration while maintaining a high level of target specificity, leading to fewer side effects. One of the examples for these is the Albumin Binding Domain-Derived Affinity Protein (ADAPT). It has been based on one of the albumin binding domains (ABD) of the streptococcal protein G, with a size of 6.5 kDa. Recently, the ADAPTs were further modified to simultaneously bind albumin and another pertinent target protein of interest, suggesting a longer half-life in patient serum, and enabling the development of newer therapeutics. This project presents the 4th generation of the ADAPT library designed to have improved stability. Phage display selections were performed against three target proteins: carcinoembryonic antigen- related cell adhesion molecule 5 (CEACAM5), a biomarker for colorectal cancer, epithelial cell adhesion molecule (EpCAM), a marker for several gastrointestinal carcinomas and trophoblast cell-surface antigen 2 (Trop2) which is overexpressed in triple-negative breast cancer. The results demonstrate binding towards CEACAM5, EpCAM and Trop2, which has been shown by monoclonal phage ELISA. The binding affinity, secondary structure of the selected binders and bispecific binding towards serum albumin remain to be further assessed. The project thus reveals that the ADAPTs selected against the targets CEACAM5, EpCAM and Trop2 present a massive potential for future clinical applications aimed towards development of diagnostics and therapeutics for these cancer biomarkers.