Untersuchung von membranadsorbersystemen in der proteinaufreinigung und entwicklung aptamer-basierter affinitätschromatografie
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- Tez No: 402649
- Danışmanlar: PROF. DR. THOMAS SCHEPER
- Tez Türü: Doktora
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: membrane adsorber, aptamer, magnetic beads
- Yıl: 2008
- Dil: Almanca
- Üniversite: Gottfried Wilhelm Leibniz Universität Hannover
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 111
Özet
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Özet (Çeviri)
Liquid chromatography is an indispensable tool in proteomics research and industrial applications. Chromatographic techniques are traditionally carried out using packed beds. The complex transport phenomena of packed bed chromatographic processes make the scale-up difficult. An effective alternative presents the membrane adsorber based downstreaming. Membrane adsorbers are composed of one or more layers of microporous and macroporous membranes, which are modified by diverse biospecific ligands. Within the scope of this thesis, a modular construction system concept based on membrane adsorber technology was developed to screen different target proteins and to devise the most efficient strategy for their fast and direct separation and purification. For the implementation of this concept, 8-strip devices were designed by Sartorius Stedim Biotech GmbH, Göttingen. Each 8-strip represents a certain membrane type (e.g., ion exchanger, metal chelate) and can be arranged in the 96-well format. To demonstrate the flexible applicability of the developed membrane adsorber based devices in a high-throughput downstream screening system, the adsorption capacity and the selectivity of the strong ion exchange (Q and S) and metal chelate (IDA) adsorber membranes, as well as the effect of loading-elution conditions, were investigated and compared with membrane modules with a larger bed volume and other types of construction (spin columns and MA 75 units). It could be shown that the binding capacity of ion exchangers is not limited by the type of construction, the expansion of the membrane bed and flow rate. However, the binding capacity was dependent on the stacks of large number of membranes, which is caused by the variable flow dispersion in the several membrane layers. An easy scale-up of the units equal number of membranes (8-strip, 15 layers, 4.8 cm² and MA 75 unit, 15 layers, 75 cm²) was demonstrated. As example of use for ion exchanger modules the human growth hormone (hGH) was succesfully purified from CHO cell culture supernatant on cation exchange membranes. The IDA 8-strips were applied in the determination of the optimal purification conditions of his tagged enzymes and the results were carried on IDA 75 units. In search of new ligands, aptamers are increasingly discussed for the application in the chromatographic purification processes. Aptamers are short single stranded DNA or RNA oligonucleotides binding their target molecules with high affinity and specificity. Therefore, in the second part of the thesis an aptamer based affinity chromatography was developed. For this purpose, two aptamers against his tag were utilised as model ligands. To immobilise the aptamers, several surfaces (porous agarose particle, membranes, magnetic beads) with different functionalities (aldehyde, epoxy, carboxyl, amino) were investigated. Under maintenance of their active conformation, the amine-modified aptamers could be immobilised on amino magnetic beads by activation with cyanuric chloride. After optimisation and characterisation of the developed aptamer chromatography, the succesful purification of his tagged proteins from fermentation broths was achieved, whereas the reusability and the longterm stability of the aptamer beads were demonstrated.
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