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Das Influenzavirus infiziert Säugetiere und Vögel. Der erste Schritt im Infektionszyklus ist die Anbindung des Viruses über sein Oberflächenprotein Hämagglutinin (HA) an Zuckerstrukturen auf Epithelzellen des respiratorischen Traktes im Wirtsorganismus. Aus den drei komplementaritätsbestimmenden Regionen (complementarity determining regions, CDRs) der schweren Kette eines monoklonalen Hämagglutinin-bindenden Antikörpers wurden drei lineare Peptide abgeleitet. Die Bindungseigenschaften der drei Peptide wurden experimentell mittels Oberflächenplasmonenresonanzspektroskopie untersucht. Es zeigte sich, dass in Übereinstimmung mit begleitenden Molekulardynamik-Simulationen zwei der drei Peptide (PeB und PeC) analog zur Bindefähigkeit des Antikörpers in der Lage sind, Influenzaviren vom Stamm X31 (H3N2 A/Aichi/2/1968) zu binden. Die Interaktion des Peptids PeB, welches potentiell mit der konservierten Rezeptorbindestelle im HA interagiert, wurde anschließend näher charakterisiert. Die Detektion der Influenzaviren war unter geeigneten Immobilisationsbedingungen im diagnostisch relevanten Bereich möglich. Die Spezifität der PeB-Virus-Bindung wurde mittels geeigneter Kontrollen auf der Seite des Analyten und des Liganden nachgewiesen. Des Weiteren war das Peptid PeB in der Lage die Bindung von X31-Viren an Mimetika seines natürlichen Rezeptors zu inhibieren, was die spezifische Interaktion mit der Rezeptorbindungsstelle im Hämagglutinin belegt. Anschließend wurde die Primärsequenz von PeB durch eine vollständige Substitutionsanalyse im Microarray-Format hinsichtlich der Struktur-Aktivitäts-Beziehungen charakterisiert. Dies führte außerdem zu verbesserten Peptidvarianten mit erhöhter Affinität und breiterer Spezifität gegen aktuelle Influenzastämme verschiedener Serotypen (z.B. H1N1/2009, H5N1/2004, H7N1/2013). Schließlich konnte durch Verwendung einer in der Primärsequenz angepassten höher affinen Peptidvariante die Influenzainfektion in vitro inhibiert werden. Damit stellen die vom ursprünglichen Peptid PeB abgeleiteten Varianten Rezeptormoleküle in biosensorischen Testsystemen sowie potentielle Wirkstoffe dar.
Plant-derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects where tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harbouring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver / reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast, than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC - EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF – DNA-binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.
COMPASS: Rapid combinatorial optimization of biochemical pathways based on artificial transcription factors
We established a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) for controlling the expression of pathway genes, and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, we equipped COMPASS with multi-locus CRISPR/Cas9-mediated modification capacity. In its current realization, COMPASS allows combinatorial optimization of up to ten pathway genes, each transcriptionally controlled by nine different ATFs spanning a 10-fold difference in expression strength. The application of COMPASS was demonstrated by generating cell libraries producing beta-carotene and co-producing beta-ionone and biosensor-responsive naringenin. COMPASS will have many applications in other synthetic biology projects that require gene expression balancing.
CaPRedit: Genome editing using CRISPR-Cas9 and plant-derived transcriptional regulators for the redirection of flux through the FPP branch-point in yeast. Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for production of different natural products. We developed CRISPR/Ca9- and plant derived regulator-mediated genome editing approach (CaPRedit) to greatly accelerate strain modification and to facilitate very low to very high expression of key enzymes using inducible regulators. CaPRedit can be implemented to enhance the production of yeast endogenous or heterologous metabolites in the yeast S. cerevisiae. The CaPRedit system aims to faciltiate modification of multiple targets within a complex metabolic pathway through providing new tools for increased expression of genes encoding rate-limiting enzymes, decreased expression of essential genes, and removed expression of competing pathways. This approach is based on CRISPR/Cas9-mediated one-step double-strand breaks to integrate modules containing IPTG-inducible plant-derived artificial transcription factor and promoter pair(s) in a desired locus or loci. Here, we used CaPRedit to redirect the yeast endogenous metabolic flux toward production of farnesyl diphosphate (FPP), a central precursor of nearly all yeast isoprenoid products, by overexpression of the enzymes lead to produce FPP from glutamate. We found significantly higher beta-carotene accumulation in the CaPRedit-mediated modified strain than in the wild type (WT) strain. More specifically, CaPRedit_FPP 1.0 strain was generated, in which three genes involved in FPP synthesis, tHMG1, ERG20, and GDH2, were inducibly overexpressed under the control of strong plant-derived ATFPs. The beta–carotene accumulated in CaPRedit_FPP 1.0 strain to a level 1.3-fold higher than the previously reported optimized strain that carries the same overexpressed genes (as well as additional genetic modifications to redirect yeast endogenous metabolism toward FPP production). Furthermore, the genetic modifications implemented in CaPRedit_FPP 1.0 strain resulted in only a very small growth defect (growth rate relative to the WT is ~ -0.03).
Nature has always served as a model for mimicking and inspiration to humans in their efforts to improve their life. Researchers have been inspired by nature to produce biomimetic materials with molecular recognition properties by design rather than evolution. Molecular imprinting is one way to prepare such materials. Such smart materials with new functionalities are at the forefront of the development of a relevant number of ongoing and perspective applications ranging from consumer to space industry. Molecularly imprinted polymers were developed by mimicking the natural enzymes or antibodies that serve as host for binding target molecules. These imprints were used as a recognition element to substitute natural biomolecules in biosensors. The concept behind molecular imprinting is to mold a material (with the desired chemical properties) around individual molecules. Upon removal of the molecular templates, one is left with regions in the molded material that fit the shape of the template molecules. Thus, molecular imprinting results in materials that can selectively bind to molecules of interest. Imprinted materials resulted in applications ranging from chemical separation to bioanalytics. In this work attempts were made particularly in the development of molecularly imprinted polymer based thermometric sensors. The main effort was focused towards the development of an covalently imprinted polymer that would be able to selectively bind fructosyl valine (Fru-Val), the N-terminal constituent of hemoglobin A1c ß-chains. Taking into account the known advantages of imprinted polymers, e.g. robustness, thermal and chemical stability, imprinted materials were successfully used as a recognition element in the sensor. One of the serious problems associated with the development of MIP sensors and which lies in the absence of a generic procedure for the transformation of the polymer-template binding event into a detectable signal has been addressed by developing the "thermometric" approach. In general the developed approach gives a new insight on MIP/Analyte interactions.
Today, analytical chemistry does not longer consist of only the big measuring devices and methods which are time consuming and expensive, which can furthermore only be handled by the qualified staff and in addition the results can also only be evaluated by this qualified staff. Usually, this technique, which shall be described in the following as 'classic analytic measuring technique', requires also rooms equipped especially and often a relative big quantity of the test compounds which should be prepared especially. Beside this classic analytic measuring technique, limited on definite substance groups and requests, a new measuring technique has gained acceptance particularly within the last years, which one can often be used by a layman, too. Often the new measuring technique has very little pieces of equipment. The needed sample volumes are also small and a special sample preparation isn't required. In addition, the new measuring instruments are simple to handle. They are cheap both in their production and in the use and they permit even a continuous measurement recording usually. Numerous of this new measuring instruments base on the research in the field of Biosensorik during the last 40 years. Since Clark and Lyon in the year 1962 were able to measure glucose with a simple oxygen electrode, completed by an enzyme the development of the new measuring technique did not have to be held back any longer. Biosensors, special pickups which consists of a combination from a biological component (permits a specific recognition of the analyte also without purification of the sample previously) and a physical pickup (convert the primary physicochemical effect into an electronically measurable signal), conquered the market. In the context of this thesis different tyrosinasesensors were developed which fulfilling the various requests, depending on origin and features of the used tyrosinase. One of the tyrosinasesensors for example was used for quantification of phenolic compounds in river and sea water and the results could correlated very well with the corresponding DIN-test for the determination of phenolic compounds. An other developed tyrosinasesensor showed a very high sensitiveness for catecholamines, substances which are of special importance in the medical diagnostics. In addition, the investigations of two different tyrosinases, which were carried out also in the context of this thesis, have shown, that a special tyrosinase (tyrosinase from Streptomyces antibioticus) will be the better choice as tyrosinase from Agaricus bisporus, which is used in the area of biosensor research till now, if one wants to develop in future even more sensitive tyrosinasesensors. Furthermore, first successes became reached on a molecular biological field, the production of tyrosinasemutants with special, before well-considered features. These successes can be used to develop a new generation of tyrosinasesensors, tyrosinasesensors in which tyrosinase can be bound directionally both to the corresponding physical pickup or also to another enzyme. From this one expects to achieve ways minimized which the substance to be determined (or whose product) otherwise must cover. Finally, this should result in an clearly visible increase of sensitivity of the Biosensor.
The aim of this thesis is the design, expression and purification of human cytochrome c mutants and their characterization with regard to electrochemical and structural properties as well as with respect to the reaction with the superoxide radical and the selected proteins sulfite oxidase from human and fungi bilirubin oxidase. All three interaction partners are studied here for the first time with human cyt c and with mutant forms of cyt c. A further aim is the incorporation of the different cyt c forms in two bioelectronic systems: an electrochemical superoxide biosensor with an enhanced sensitivity and a protein multilayer assembly with and without bilirubin oxidase on electrodes. The first part of the thesis is dedicated to the design, expression and characterization of the mutants. A focus is here the electrochemical characterization of the protein in solution and immobilized on electrodes. Further the reaction of these mutants with superoxide was investigated and the possible reaction mechanisms are discussed. In the second part of the work an amperometric superoxide biosensor with selected human cytochrome c mutants was constructed and the performance of the sensor electrodes was studied. The human wild-type and four of the five mutant electrodes could be applied successfully for the detection of the superoxide radical. In the third part of the thesis the reaction of horse heart cyt c, the human wild-type and seven human cyt c mutants with the two proteins sulfite oxidase and bilirubin oxidase was studied electrochemically and the influence of the mutations on the electron transfer reactions was discussed. Finally protein multilayer electrodes with different cyt form including the mutant forms G77K and N70K which exhibit different reaction rates towards BOD were investigated and BOD together with the wild-type and engineered cyt c was embedded in the multilayer assembly. The relevant electron transfer steps and the kinetic behavior of the multilayer electrodes are investigated since the functionality of electroactive multilayer assemblies with incorporated redox proteins is often limited by the electron transfer abilities of the proteins within the multilayer. The formation via the layer-by-layer technique and the kinetic behavior of the mono and bi-protein multilayer system are studied by SPR and cyclic voltammetry. In conclusion this thesis shows that protein engineering is a helpful instrument to study protein reactions as well as electron transfer mechanisms of complex bioelectronic systems (such as bi-protein multilayers). Furthermore, the possibility to design tailored recognition elements for the construction of biosensors with an improved performance is demonstrated.
Electron transfer (ET) reactions play a crucial role in the metabolic pathways of all organisms. In biotechnological approaches, the redox properties of the protein cytochrome c (cyt c), which acts as an electron shuttle in the respiratory chain, was utilized to engineer ET chains on electrode surfaces. With the help of the biopolymer DNA, the redox protein assembles into electro active multilayer (ML) systems, providing a biocompatible matrix for the entrapment of proteins.
In this study the characteristics of the cyt c and DNA interaction were defined on the molecular level for the first time and the binding sites of DNA on cyt c were identified. Persistent cyt c/DNA complexes were formed in solution under the assembly conditions of ML architectures, i.e. pH 5.0 and low ionic strength. At pH 7.0, no agglomerates were formed, permitting the characterization of the NMR spectroscopy. Using transverse relaxation-optimized spectroscopy (TROSY)-heteronuclear single quantum coherence (HSQC) experiments, DNAs’ binding sites on the protein were identified. In particular, negatively charged AA residues, which are known interaction sites in cyt c/protein binding were identified as the main contact points of cyt c and DNA.
Moreover, the sophisticated task of arranging proteins on electrode surfaces to create functional ET chains was addressed. Therefore, two different enzyme types, the flavin dependent fructose dehydrogenase (FDH) and the pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH), were tested as reaction partners of freely diffusing cyt c and cyt c immobilized on electrodes in mono- and MLs. The characterisation of the ET processes was performed by means of electrochemistry and the protein deposition was monitored by microgravimetric measurements. FDH and PQQ-GDH were found to be generally suitable for combination with the cyt c/DNA ML system, since both enzymes interact with cyt c in solution and in the immobilized state. The immobilization of FDH and cyt c was achieved with the enzyme on top of a cyt c monolayer electrode without the help of a polyelectrolyte. Combining FDH with the cyt c/DNA ML system did not succeed, yet. However, the basic conditions for this protein-protein interaction were defined. PQQ-GDH was successfully coupled with the ML system, demonstrating that that the cyt c/DNA ML system provides a suitable interface for enzymes and that the creation of signal chains, based on the idea of co-immobilized proteins is feasible.
Future work may be directed to the investigation of cyt c/DNA interaction under the precise conditions of ML assembly. Therefore, solid state NMR or X-ray crystallography may be required. Based on the results of this study, the combination of FDH with the ML system should be addressed. Moreover, alternative types of enzymes may be tested as catalytic component of the ML assembly, aiming on the development of innovative biosensor applications.