Design and synthesis of submicron polymer particles for the selective capture of proteases
FacultiesFakultät für Naturwissenschaften
InstitutionsInstitut für Analytische und Bioanalytische Chemie
Institut für Organische Chemie III (Makromolekulare Chemie und Organische Materialien)
The design of biomimetic synthetic receptors capable of mimicking the molecular recognition properties of antibodies is of significant interest for the application in many analytical methods. The selective binding of proteases is of great importance for the application in the purification process of pharmaceutical proteins, as proteases are frequently obtained as side product during their production and may degrade these. This thesis presents two approaches for the design and synthesis of a synthetic receptor material based on submicron polymer particles, which can bind pepsin, an example protease, with high affinity and selectivity. In the first approach the protein surface imprinting is used. This is a technique of the molecular imprinting and enables the formation of specific recognition sites on the surface of synthetic polymers through the use of a template molecule. Pepsin surface imprinted submicron polymer particles are prepared via miniemulsion polymerization. Different acrylates, methacrylic acid and an acrylamide are used as functional monomers, ethylene glycol dimethacrylate is used as cross-linker in a molar ratio of 1 : 4 and water as continuous phase. The amount of functional monomer, the pepsin template amount, pH value and type of extraction solution are varied. Furthermore, the influence of the surfactant is investigated. The pepsin binding capacity, affinity, selectivity and the imprinting efficiency are evaluated with single and competitive batch rebinding experiments with pepsin and different other proteins using UV-Vis spectrophotometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the second approach the type and ratio of acrylamides with different functional groups are selected according to the chemical composition of the surface of pepsin and varied to obtain the highest possible number of interactions between the resulting polymer particles and pepsin. Lightly cross-linked polymer nanoparticles are prepared with a low monomer concentration and a low amount of surfactant in water via precipitation polymerization. The polymer particles with different compositions are screened for the one with the highest affinity to pepsin. This is obtained with an incubation study using UV-Vis-spectrophotometry and an incubation study in microtiter wells using a protease assay for a fast screening and the direct detection of bound pepsin.
Subject HeadingsPepsin [GND]
Proteolytic enzymes [LCSH]