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V magistrskem delu je predstavljen razvoj monolitnih kromatografskih kolon majhnih volumnov (v nadaljevanju mini diski), optimiranih za afinitetno kromatografijo. Razvito je bilo 3-delno ohišje iz polioksimetilena kopolimera (POM-C), ki omogoča uporabo mini diskov treh različnih velikostnih razredov (50 µL, 100 µL in 200µL) tako na HPLC/FPLC sistemih kot tudi brez – z ročnim prečrpavanjem preko brizg. Optimizacija ohišja je vključevala manjšanje mrtvega volumna in odpravo nespecifičnih adsorbcij bioloških molekul na kromatografske frite. Dokaz uspešne priprave afinitetnih mini diskov smo dosegli z imobilizacijo rekombinantnega proteina A na stacionarno fazo. Z uvedbo predhodne kvalitativne karakterizacije mini diskov v fazi hidroksi modifikacije monolita, smo s testom pulzne motnje (testiranje puščanja) in uvedbo benzimidazolne dinamične vezavne kapacitete, kot analoga IgG (testiranje homogenosti), dosegli optimizacijo imobilizacije in celotnega procesa monolitne afinitetne kromatografije. Pripravo encimskega reaktorja smo dosegli z izolacijo encima proteaza TEV iz bakterije Escherichia coli, katerega smo kovalentno vezali na različno aktivirane (karbonil diimidazol (CDI) in aldehidne skupine) mini diske. Aktivnost vezane proteaze TEV smo preverjali s cepitvijo afinitetnega podaljška (His-repek) rekombinantnih proteinov Listeriolizin O (LLO) in DARPina. Cepitve afinitetnih podaljškov pri rekombinantnih proteinih z uporabo encimskih reaktorjev nismo dosegli v nobenem primeru, kljub dokazani encimski aktivnosti proteaze TEV za cepitev DARPina v raztopini. Za dosego uspešnosti cepitve, bi bila potrebna nadalnja raziskovanja v smeri optimizacije imobilizacije (vezava encima z uporabo aldehidne ročice, ali disulfidna kovalenta vezava preko tiolnih skupin cisteinskih ostankov) ter testiranje aktivnosti encima v različnih imobilizacijskih pufrskih raztopinah. Development of monolithic chromatographic columns with small volumes (in continuation: mini discs) in presented in this master's thesis. A 3-part chromatographic housing made from polyoxymethylen copolimer (POM-C) was developed. It enables the usage of three different size-classes of mini discs (50µL, 100 µL and 200 µL), which can be used either on HPLC or FPLC systems, or manually via the attachment on syringe. The optimization of housing was achieved by reducing its dead volume and non-specific binding adsorption of bio-molecules on chromatographic frits. Demonstration of successful preparation of affinity mini disc was achieved by immobilization of a recombinant protein A on stationary phase (monolith). With the implementation of preliminary qualitative characterization of mini discs in the initial development phase – hydroxy modification, such as pulse response (integrity test) and benzimidazole dynamic binding capacity as analog of IgG (test of homogeneity), we achieved optimization of immobilization and the entire process of monolith affinity chromatography preparation. Preparation of immobilized enzyme reactor was achieved by covalent immobilization of TEV protease that was previously isolated from Escherichia coli. The immobilization was performed on various activated mini discs carriers (carbonyl diimidazole (CDI) and aldehyde groups). Activity of TEV protease was examined by cleaving the affinity histidine tag from recombinant proteins Listeriolysine O and DARPin. The cleavage of his-tag on mini discs IMERs with TEV protease was not successful, in spite of successful cleavage of His- tagged DARPin obtained by TEV protease in solution. In order to achieve successful cleavage, further optimization of is required (covalent binding using an aldehyde spacer, or disulfide covalent binding via thiol groups of cysteine residues), as well as stability and activity testing of the enzyme in various immobilization buffer solutions. |
