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For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 µg protein is used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 µg) immobilised on metal chelate resins. The screen was successfully applied to fold a human protein, the p22 subunit of dynactin that is expressed in inclusion bodies in E. coli. For p22 dynactin – as is the case for many proteins – there was no biological assay available to assess the success of the folding screen. Protein solubility can not be used as a stringent criterion because beside natively folded protein, soluble misfolded species and microaggregates may occur. This work evaluates methods to detect small amounts of natively folded protein after automated folding screening. Before folding screening with p22 dynactin, two model enzymes, bovine carbonic anhydrase II (CAB) and pig heart mitochondrial malate dehydrogenase, were used for evaluation. Recovered activity after refolding was correlated to different biophysical methods. 8-anilino-1-naphtalenesulfonic acid binding-experiments gave no useful information when refolding CAB, due to low sensitivity and because misfolded protein could not be readily distinguished from native protein. Tryptophan fluorescence spectra of refolded CAB were used to assess the success of refolding. The shift of the intensity maximum to a shorter wavelength, compared to the denaturant unfolded protein, as well as the fluorescence intensity correlated to recovered enzymatic activity. For both model enzymes, analytical hydrophobic interaction chromatography (HIC) was useful to identify refolded samples that contain active enzyme. Compactly folded, active enzyme eluted in a distinct peak in a decreasing ammonium sulfate gradient. The detection limit of analytical HIC was approx. 5 µg. In case of CAB, tryptophan fluorescence spectroscopy and analytical HIC showed that both methods in combination can be useful to rule out false positives or false negatives obtained with one method. These two methods were also useful to identify conditions for folding of p22 dynactin. However, tryptophan fluorescence spectroscopy can lead to false positives because in some cases spectra of soluble microaggregates are not well distinguishable from spectra of natively folded protein. In summary, a fast and reliable screening procedure was developed to make inclusion body proteins accessible to structural or functional analyses. In a separate project, 88 different E. coli expression constructs for 17 human protein domains that had been identified by sequence analysis were analysed using high-throughput purification and folding analysis in order to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, solubly expressed protein domains were directly analysed using 1D ¹H-NMR spectroscopy. It was found that isolated methyl group signals below 0.5 ppm are particularly sensitive and reliable probes for folded protein. In addition – similar to the evaluation of a folding screen – analytical HIC proved to be an efficient tool for identifying constructs that yield compactly folded protein. Both methods, 1D ¹H-NMR spectroscopy and analytical HIC, provided complementary results. Six constructs, representing two domains, could be quickly identified as targets that are well suitable for structural analysis. The structure of one of these domains was solved recently by co-workers, the other structure was published by another group during this project.
Recent high-throughput technologies enable the acquisition of a variety of complementary data and imply regulatory networks on the systems biology level. A common approach to the reconstruction of such networks is the cluster analysis which is based on a similarity measure. We use the information theoretic concept of the mutual information, that has been originally defined for discrete data, as a measure of similarity and propose an extension to a commonly applied algorithm for its calculation from continuous biological data. We compare our approach to previously existing algorithms. We develop a performance optimised software package for the application of the mutual information to large-scale datasets. Furthermore, we design and implement a web-based service for the analysis of integrated data measured with different technologies. Application to biological data reveals biologically relevant groupings and reconstructed signalling networks show agreements with physiological findings.
The development of fast and reliable biochemical tools for on-site screening in environmental analysis was the main target of the present work. Due to various hazardous effects such as endocrine disruption and toxicity phenolic compounds are key analytes in environmental analysis and thus were chosen as model analytes. Three different methods were developed: For the enzymatic detection of phenols in environmental samples an enzyme-based biosensor was developed. In contrast to reported work using tyrosinase or peroxidases, we developed a biosensor based on glucose dehydrogenase as biorecognition element. This biosensor was devoted for an application in a laboratory flow system as well as in a portable device for on-site measurements. This enzymatic detection is applicable only for a limited number of phenols due to substrate specificity of the enzyme. For other relevant compounds based on a phenolic structure (i.e. nitrophenol, alkylphenols and alkylphenol ethoxylates) immunological methods had to be developed. The electrochemical GDH-biosensor was used as the label detector in these immunoassays. Two heterogeneous immunoassays were developed where ßGal was used as the label. An electrochemical method for the determination of the marker enzyme activity was processed. The separation step was realized with protein A/G columns (laboratory flow system) or by direct immobilization of the antibodies in small disposable capillaries (on-site analysis). All methods were targeted on the contemporary analysis of small numbers of samples.
Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent. To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique. Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features. These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.
In this thesis, I investigated the factors influencing the growth and vertical distribution of planktonic algae in extremely acidic mining lakes (pH 2-3). In the focal study site, Lake 111 (pH 2.7; Lusatia, Germany), the chrysophyte, Ochromonas sp., dominates in the upper water strata and the chlorophyte, Chlamydomonas sp., in the deeper strata, forming a pronounced deep chlorophyll maximum (DCM). Inorganic carbon (IC) limitation influenced the phototrophic growth of Chlamydomonas sp. in the upper water strata. Conversely, in deeper strata, light limited its phototrophic growth. When compared with published data for algae from neutral lakes, Chlamydomonas sp. from Lake 111 exhibited a lower maximum growth rate, an enhanced compensation point and higher dark respiration rates, suggesting higher metabolic costs due to the extreme physico-chemical conditions. The photosynthetic performance of Chlamydomonas sp. decreased in high-light-adapted cells when IC limited. In addition, the minimal phosphorus (P) cell quota was suggestive of a higher P requirement under IC limitation. Subsequently, it was shown that Chlamydomonas sp. was a mixotroph, able to enhance its growth rate by taking up dissolved organic carbon (DOC) via osmotrophy. Therefore, it could survive in deeper water strata where DOC concentrations were higher and light limited. However, neither IC limitation, P availability nor in situ DOC concentrations (bottom-up control) could fully explain the vertical distribution of Chlamydomonas sp. in Lake 111. Conversely, when a novel approach was adopted, the grazing influence of the phagotrophic phototroph, Ochromonas sp., was found to exert top-down control on its prey (Chlamydomonas sp.) reducing prey abundance in the upper water strata. This, coupled with the fact that Chlamydomonas sp. uses DOC for growth, leads to a pronounced accumulation of Chlamydomonas sp. cells at depth; an apparent DCM. Therefore, grazing appears to be the main factor influencing the vertical distribution of algae observed in Lake 111. The knowledge gained from this thesis provides information essential for predicting the effect of strategies to neutralize the acidic mining lakes on the food-web.
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.