24 research outputs found

    An investigation into approaches and pathways relevant to cellular flocculation in CHO cells

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    Over the last two decades, viable cell concentrations in industrial processes have reached multiples of 107 cells/mL at harvest. Given that many production facilities were built to handle viable cell concentrations in the order of 106 cells/mL, cell concentrations of this magnitude have the potential to cause problems during primary recovery activities as existing processes/equipment may not be able to efficiently remove cells from these high cell concentration processes. Flocculation, the ability of cells to clump together is one solution to this issue. The objective of this thesis was to engineer a flocculating CHO cell line. Three distinct approaches are described. The first approach was the cloning, in CHO cells of four eukaryotic proteins known to promote flocculation. The second approach used inorganic cations as flocculating agents and the third approach utilised proteomics to investigate cellular adhesion in CHO cells. The nature of the proteins investigated in this thesis lead to challenges in achieving ectopic expression in CHO cells. While the addition of FeCl3, MnCl2 and CaCl2 did result in improved rates of sedimentation of CHO cells compared to untreated controls without any negative impact on a human antibodies structural integrity, the antibody yield at 62% for MnCl2,72% FeCl3 and 76% for CaCl2 was not commercially viable. A differential expression proteomic approach identified a cohort of proteins involved in the catabolism of amino acids, the catabolism of fatty acids, cholesterol metabolism, protein processing in the endoplasmic reticulum and glycolysis/gluconeogenesis which were significantly enriched following the adaptation from attached to suspension growth. Additionally, the thesis proposes a potential mechanism which CHO cells developed to combat increased cellular stress during this adaptation process. This thesis is an describes the first steps towards engineering a flocculating CHO cell line suitable for use with current commercial processes

    A Novel SCP-RICM Assay Application: Indirect Detection of Analytes by Modulation of Protein-Protein Interactions

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    The SCP-RICM assay employs the measurable surface energy (or adhesive work W_adh) of a micrometer-sized polymeric sphere (soft colloidal probe, SCP) interacting with a glass chip using reflection interfer-ence contrast microscopy (RICM). Depending on those two interacting surfaces' nature and functional-ization, the SCP will deform, creating a contact area with the hard glass chip. This contact area is clearly distinguishable from the sphere’s interference ring pattern and can be measured. The adhesive surface energy W_adh can be calculated from the size of the contact area. An immobilization can be overcome by choosing a two-component analyte-dependent interaction, here presented for the copper (Cu) detection. The detection of Cu was chosen as a proof-of-concept system. However, detecting metal ions is an essential endeavor because, in excessive amounts, they present a severe threat to health and the environment. The copper-dependent interaction of the yeast chaperones yCox17 (also Cox17) and ySco1 (also Sco1) were chosen as the two-component analyte-dependent interaction. The chaperones partic-ipate in vivo in the formation of the electron transport chain of S. cerevisiae and interact in the mito-chondrial inner membrane to transfer one Cu(I) ion from Cox17 to Sco1. It was necessary to immobilize one protein to the SCPs and one to the chip surface, to transfer the copper chaperones' interaction into the SCP-RICM assay core detection components. The unique self-assembling characteristics of the class I hydrophobin Ccg-2 from N. crassa were used to immobilize one interaction partner to the chip surface. Class I hydrophobins are known for the formation of re-sistant and uniform layers at hydrophilic/hydrophobic interfaces. Initial SCP-RICM assay measurements with Sco1Δ95_a-SCPs and the Cox17_c-chips indicate that copper detection using the proposed mechanism is possible (Figure 39-3). Measurements can be differentiated between 0 and 0.1 mM Cu(I) concentration in solution. Further screening of concentrations be-low 0.1 mM is still necessary. The presented proof-of-principle system for the indirect detection of copper shows copper-dependent behavior. These positive results give rise to many more options to use the SCP-RICM assay as an indirect detection system. The application range of the SCP-RICM assay could be enlarged for different analytes such as other heavy metals, bacteriophages, biomarkers, et cetera, and is relevant for fields from medicine to environmental monitoring.:TABLE OF CONTENT Table of Content I List of Figures VII List of Tables IX List of Abbreviations XI 1 Introduction 1 1.1 Biosensors 1 1.2 Analytical Detection Methods: Copper 2 1.3 SCP-RICM Assay 3 1.3.1 Sensor Chip Surface 4 1.3.2 Soft Colloidal Probes 5 1.3.3 Reflection Interference Contrast Microscopy 6 1.4 Hydrophobins 9 1.4.1 Structure and Functions of Hydrophobins 9 1.4.2 Ex vivo Applications of Hydrophobins 11 1.4.3 Class I Hydrophobin: Ccg-2 12 1.5 Mitochondrial Respiratory Chain 14 1.5.1 Copper Transport in Yeast 14 1.5.2 S. cerevisiae Sco1 protein 18 1.5.3 S. cerevisiae Cox17 protein 21 1.6 SCP-RICM Assay for Copper Detection 23 1.7 Aim of the Study 24 2 Materials and Methods 25 2.1 Laboratory Equipment 25 2.1.1 Devices 25 2.1.2 Chemicals 26 2.1.3 Consumables 28 2.1.4 Antibodies 29 2.1.5 Enzymes 30 2.1.6 Molecular Weight Standards 30 2.1.7 DNA Oligonucleotides 31 2.1.8 Plasmids and Vectors 32 2.2 Microorganisms 33 2.2.1 Strains 33 2.2.2 Cultivation of Microorganisms 34 2.2.3 Preparation of Electrocompetent E. coli Cells 36 2.2.4 Preparation of E. coli Glycerol Stocks 36 2.3 Protein Design 37 2.4 Molecular Cloning Methods 38 2.4.1 Vector Template Preparation 38 2.4.2 Agarose Gel Electrophoresis 40 2.4.3 DNA Extraction from Agarose Gels 41 2.4.4 Polymerase Chain Reaction 41 2.4.5 DNA Restriction Digest 42 2.4.6 DNA Dialysis 43 2.4.7 Ligation of DNA Fragments 43 2.4.8 Isolation of DNA from E. coli 44 2.4.9 DNA Sequencing 45 2.4.10 Transformation of E. coli via Electroporation 45 2.5 Protein Detection and Quantification 46 2.5.1 SDS PAGE 46 2.5.2 Coomassie Staining 50 2.5.3 Western Blot Analysis 51 2.5.4 Immunological Detection 51 2.5.5 Protein Quantification: Lowry Assay 52 2.5.6 Protein Quantification: Bradford Assay 53 2.5.7 Protein Quantification: NanoDrop Measurement 53 2.6 Protein Purification and Storage 54 2.6.1 Expression Analysis of Recombinant Proteins 54 2.6.2 Solubility Analysis 54 2.6.3 Protein Purification by Ni2+ Affinity Chromatography 55 2.6.4 Quantification of Purified Proteins 64 2.6.5 Dialysis of Purified Proteins 65 2.7 Glass Surface Functionalization 65 2.7.1 Glass Surface Preparation 66 2.7.2 Hydrophobin and Fusion Protein-Based Coating 66 2.7.3 Contact Angle Measurement 67 2.7.4 DRoPS Test 67 2.7.5 Atomic Force Microscopy 67 2.8 SCP Functionalization 68 2.8.1 Functionalization of SCPs with Proteins 68 2.8.2 Validation of SCP Functionalization with FITC Staining 69 2.9 SCP-RICM Assay and Its Analysis 69 3 Results 73 3.1 Generation of Recombinant Fusion Proteins 73 3.1.1 Sco1 and Sco1∆95 73 3.1.2 Cox17 84 3.1.3 Ccg-2 88 3.1.4 Overview: Optimization of Expression and Purification of Recombinant Proteins 90 3.2 His-Tag Cleavage 92 3.3 Chip Surface Functionalization 94 3.3.1 Optimization of the Glass Chip Preparation 94 3.3.2 Macroscopic Properties of the Functionalized Chip Surface 95 3.3.3 AFM Measurements 102 3.3.4 Theoretical Package of Hydrophobin Ccg-2 on the Chip Surface 103 3.4 SCP Functionalization 104 3.4.1 SCP Functionalization and FITC Staining 104 3.4.2 Theoretical Package of Proteins on SCPs 106 3.5 SCP-RICM Assay 107 4 Discussion and Further Prospectives 113 4.1 Discussion: SCP-RICM Assay and Protein-Protein Interaction 113 4.2 Outlook and Further Prospects 119 4.2.1 Heterologous Protein Expression and Purification: Methods, Cleavage and Refolding 119 4.2.2 Further Analysis of Chip Surface Functionalization 124 4.2.3 Alternative Chip Surface Functionalization Methods 126 4.2.4 SCP-RICM Assay: Data Acquisition and Evaluation 128 4.2.5 SCP-RICM Assay: Copper Detection 130 4.2.6 Exploiting the SCP-RICM Assay using Protein-Protein Interactions 131 4.2.7 Exploiting the SCP-RICM Assay with Alternative Interactions 133 5 Summary 137 6 Bibliography 141 7 Appendix 165 7.1 Sequences of Protein Constructs 165 7.1.1 Sequences of the Protein Construct Cox17_a 165 7.1.2 Sequences of the Hydrophobin-Cox17 Fusion Protein Cox17_b 165 7.1.3 Sequences of the Hydrophobin-Cox17 Fusion Protein Construct Cox17_c 166 7.1.4 Sequences of the Protein Construct Sco1_a and Sco1Δ95_a 167 7.1.5 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_b and Sco1Δ95_b 169 7.1.6 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_c and Sco1Δ95_c 171 7.1.7 Sequences of the Hydrophobin Ccg-2 173 7.2 pET-28b(+): Plasmid Map 173 7.3 Nickel Removal During Dialysis 175 7.4 DGR Assay 176 7.5 SCP diameter 179 Acknowledgements 181 Declaration of Authorship 18

    Formation, flow and dynamic stability of foams generated from viscous shear-thinning fluids

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    Foams are complex, multi-component, structures that are present in a wide range of industrial applications such as foods, pharmaceuticals, mineral transport, oil and gas. Specifically, in the food industry, foaming is ubiquitous since many foamed products, for example ice cream, whipped cream and chocolate mousse contain air in the form of microscopic bubbles. These bubbles reduce the number of calories, impart better texture and improve organoleptic properties. Therefore, a foaming operation which can achieve a good degree of control of the air volume fraction and bubble size distribution is of paramount importance. In this research study, an advanced, non-invasive, X-ray micro-Computed Tomography technique is adopted to probe the three-dimensional microstructure of a range of wet foams generated from viscous pseudoplastic fluids using a pilot-scale continuous rotor-stator device. For the first time ever, an extensive study is conducted to elucidate the combined influence of processing parameters (rotor speed and gas-liquid volumetric flowrate (G/L) ratio) and liquid properties (surfactant content and xanthan gum concentration) on foam formation from shear-thinning fluids using a multi-stage rotor-stator unit. Rotor speed, residence time and G/L ratio were the dominant factors responsible for achieving fine-textured and highly statically stable foams. In addition, operating at N > 2000 rpm is undesirable both in terms of energy efficiency and product microstructure. The dynamic foam stability is investigated by passing wet foams through narrow orifice constrictions. It is established that the microstructure of wet foams is preserved when allowed to flow through a narrow orifice constriction provided there is a minimal pressure drop ( 36000 Pa), however, a combination of foam expansion and bubble coalescence is responsible for the loss of air volume and bubble coalescence. Furthermore, by increasing the rotor speed (i.e. energy input), surfactant concentration and continuous phase apparent viscosity, the rate of bubble coalescence can be significantly reduced. The effects of rotor speed, however, became only significant at a rotor speed of N = 2000 rpm. Such a wet foam, with a more uniform bubble size distribution, was able to pass through a narrow orifice constriction with a relatively less bubble coalescence. An extensive study of steady shear and dynamic oscillatory rheometry of wet foams generated from viscous shear-thinning fluids is also conducted as part of this research. At air volume fractions of 0.60, the apparent viscosity and storage moduli increase significantly as bubble size becomes smaller and more uniform with increasing rotor speed. Moreover, a novel simultaneous in-situ foam microstructure visualisation and rheometry are conducted to explicate the flow complexities that can arise when such structured fluids are imposed to higher shear rates. Simultaneous in-situ visualisation of the foams under shear confirmed the absence of bubble breakage and, for the first time, unravelled the existence of inward radial shear-induced migration of liquid, which is responsible for the time-dependence of the foams

    Hydrophobins: Biological application of fungal proteins

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    Hydrophobin protiens are unique to the fungal kingdome and have evolved to function in different roles during the growth and development of filamentous fungi. Due to the unique properties of these proteins to self-assemble into amphipathic monolayers at hydrophobic:hydrophlic interfaces, they can be found as biosurfactants, protective coatings and as primers to enhance surface adhesion. In recent decades there has been a significant development towards the applying these proteins to a range of different research fields, from food technology and surface coatings to drug delivery devices. However, the understanding of the mechanism in which these proteins undergo self-assemble at the interface is still lacking. In this project, I have combined high resolution imagaing techniques, such as AFM, TEM and TEM tomography to compare the substructure of different Class I hydrophobin rodlet films, and using colometric kinetic assays to delineate a model for the assembly mechanisms at the interface. With the information, I was able to reveal that the exposure of hydrophobin proteins to the surface interface is a determining factor. By altering the surface interface with additives, such as ethanol, it was possible to manipulate the hydrophobin film structure and physioproperties. This knowledge was used to successfully formulate a nanosupsension of hydrophobin with hydrophobic compounds, such as curcumin and Amphotericin B. This research project lays the foundation for the future development and refinement of hydrophobin-based technologies

    Expression und Charakterisierung von Aspergillus nidulans Hydrophobinen und deren potentieller Einsatz im Denkmalschutz

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    Pilzliche Hydrophobine sind kleine, amphiphile Proteine, die sich an hydrophil-hydrophoben GrenzflĂ€chen zu einem Monolayer assemblieren und so die OberflĂ€cheneigenschaften verĂ€ndern, was sie fĂŒr eine Vielzahl an biotechnologischen Anwendungen interessant macht. In dieser Arbeit wurde ein Expressionssystem entwickelt, das die heterologe Expression von Hydrophobinen unterschiedlicher Klasse in Escherichia coli ohne die Fusion an einen permanenten N-terminalen Tag ermöglicht. Durch den Einsatz des Signalpeptids PelB war es möglich, die Hydrophobine DewA, DewC, DewD und DewE aus Aspergillus nidulans und HFBI aus Trichoderma reesei zu produzieren und ihre biochemischen Eigenschaften zu charakterisieren. Alle Hydrophobine waren in der Lage an OberflĂ€chen zu binden, die HydrophobizitĂ€t von Glas zu erhöhen und zeigten emulgatorische FĂ€higkeiten in einem Öl-Wasser Gemisch. WĂ€hrend das typische Klasse I Hydrophobin DewA die stabilsten Beschichtungen auf harten OberflĂ€chen bildete, zeigten die nicht direkt einer Klasse zuordbaren Hydrophobine DewD und DewE die besten Eigenschaften als Emulgator. Die Ergebnisse unterstĂŒtzen die zuletzt aufgekommenen Forderungen nach einer Überarbeitung der Einteilung der Hydrophobine und postulieren die EinfĂŒhrung einer neuen, intermediĂ€ren Klasse. ZusĂ€tzlich wurde der Einsatz der Hydrophobine DewA und HFBI im Denkmalschutz untersucht. Die Proteine waren in der Lage, eine hydrophobe aber Wasserdampf-durchlĂ€ssige Beschichtung nach dem GoreTexÂź-Modell auf drei unterschiedlichen Lithotypen zu bilden. Die große Eindringtiefe der Hydrophobine in das Gestein macht einen Einsatz jedoch nicht nur als wasserabweisende Beschichtung, sondern auch als Vorbehandlung bei der Konsolidierung von brĂŒchigem Gestein denkbar. Die Eigenschaft, die PolaritĂ€t von OberflĂ€chen zu verĂ€ndern und die OberflĂ€chenspannung von FlĂŒssigkeiten zu verringern, könnte zu einem besseren Eindringen der Konsolidierungsmittel in den Stein fĂŒhren. Abschließend wurden antimikrobielle Peptide an das Hydrophobin DewA fusioniert, heterolog aus E. coli und Saccharomyces cerevisiae aufgereinigt und auf ihre antibakterielle und fungizide Wirkung untersucht. Die hergestellten bakteriziden OberflĂ€chen sind ein erster Schritt im Kampf gegen Antibiotika-resistente BakterienstĂ€mme

    Nanocellulose from the Appalachian Hardwood Forest and Its Potential Applications

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    Nanofibrillated cellulose (NFCs) are nanoscale fibers of high aspect ratio that can be isolated from a wide variety of cellulosic sources, including wood and bacterial cellulose. With high strength despite of their low density, NFCs are a promising renewable building block for the preparation of nanostructured materials and composites. To fabricate NFC-based materials with improved mechanical and chemical properties and additional new functionalities for different applications, it is essential to tailor the surface properties of individual NFCs. The surface structures control the interactions between NFCs and ultimately dictate the structure and macroscale properties of the bulk material. This research was focused on determining the feasibility of using hardwood residues from the Appalachian Hardwood Forest for the production of nanofibrillated cellulose (NFC). In addition, some modifications during the NFC production process were performed to evaluate their improvement to incorporate more antimicrobial copper in the cellulosic backbone. This thesis has been divided in the following main chapters: 1) Literature review regarding to nanocellulosic materials and their production processes, 2) Nanocellulose current and potential applications, 3) Nanofibrillated cellulose from the Appalachian Hardwood logging residues, 4) Modified nanofibrillated from the Appalachian Hardwood logging residues, 5) Preparation of nanocellulose using ionic liquids -- A review, 6) Nanocellulose-based drug delivery system -- A review, 7) Safety aspects on the utilization of lignocellulosic based materials - A review

    Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications

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    Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application

    Molecular dynamics simulations of protein adsorption at interfaces

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    Proteins can often adsorb irreversibly at fluid/fluid interfaces; the understanding of the adsorption mechanism has relevance across a variety of industrial (e.g. the creation of stable emulsions) and biological (e.g. biofilm formation) processes. I performed molecular dynamics simulations of two surfactant proteins as they interact with air/water and oil/water interfaces, describing the origin of the surface activity, the adsorption dynamics and the conformational changes that these proteins undergo at the interface. BslA is an amphiphilic protein that forms a highly hydrophobic coat around B. subtilis biofilms, shielding the bacterial community from an external aqueous solution. By investigating the behaviour of BslA variants at oil/water interfaces via coarse-grained molecular dynamics, I show that BslA represents a biological example of an ellipsoidal Janus nanoparticle, whose surface interactions are controlled by a local conformational change. All-atom molecular dynamics simulations then reveal the details of the conformational change of the protein upon adsorption, and the self-assembly into a two-dimensional interfacial crystal. Ranaspumin-2 is one of the main components of the tungara frog foam nest. Contrary to most surfactant proteins, its structure lacks any sign of amphiphilicity. All-atom simulations show that the adsorption proceeds via a two-step mechanism where firstly the protein binds to the interface through its flexible N-terminal tail and then it undergoes a large conformational change in which the hydrophobic core becomes exposed to the oil phase. I then developed a simple structure-based coarse-grained model that highlights the same adsorption mechanism observed in all-atom simulations, and I used it to compare the dynamics of adsorption and the underlying free energy landscape of several mutants. These results agree with and are used to rationalise the observations from Langmuir trough and pendant drop experiments. Colloids can often be considered simpler versions of proteins that lack conformational changes. I performed coarse-grained simulations of the compression of interfacial monolayers formed by rod-like particles. These simulations show a rich behaviour characterised by the flipping of adsorbed rods, nematic ordering and bilayer formation. I report the series of transitions that take place as the rod aspect ratio is increased from 3 to 15
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