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Magnetic enzyme reactor with proteinase K prepared for prion protein digestion.
Authors: Peyrin Jean Michel | Le Nel Anne | Minc Nicolas | Taverna Myriam | Bílková Zuzana | Viovy Jean-Louis
Year: 2005
Type of publication: ostatní - přednáška nebo poster
Name of source: 11th International Symposium on Separation Science (ISSS 2005)
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Language Name Abstract Keywords
cze Magnetický enzymový reaktor s proteinázou K připravený pro průkaz prionového proteinu. Enzym proteináza K byl imobilizován kovalentní vazbou na magnetický nosič, metodou karbodiimidové aktivace a byl použit ke štěpení modelového proteinu Somatropinu. Enzymový reaktor byl použit také ke štěpení a průkazu prionového proteinu. proteinase K; microfluididní zařízení; magnetické partikule; enzymový reaktor
eng Magnetic enzyme reactor with proteinase K prepared for prion protein digestion. As an improvement for determination of the scrapie form of Prion protein (PrPSc), the utilization of immobilized proteinase K is described. Proteinase K was immobilized on magnetic latex nanoparticles functionalised by carboxylic groups. Several immobilization conditions and method of specific activity determination were investigated [1,2], including concentrations of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (sulfo-NHS), and proteinase K (PK), the reaction buffer type, pH, temperature, and concentration of added Ca2+ ions. Particular immobilization steps were controlled by measurement of potential of the particles. Enzyme activities of proteinase K reactors were observed and compared to the features of the free enzyme and of commercial non-magnetic proteinase K acrylic beads. The optimum immobilization chemistry consisted of 0.079 mM EDC, 12 mM sulfo-NHS, 10 mM sodium phosphate buffer at a pH of 7.3, and 2-3 mg/ml PK. Use of magnetic enzyme reactor confirmed high comfort during lab work. Magnetic proteinase K particles were used for digestion of the recombinant human growth hormone and the Prion protein. The protein digestion was performed in a microfluidic device fabricated in polydimethylsiloxane (PDMS) by soft lithography and rapid prototyping [3]. A model protein was then digested in the microfluidic channel and analytical techniques such as SDS-PAGE or CE were employed to assess proteolytic digestion efficiency. The influence of different flow rates and residence times of the protein solution on the digestion was investigated. Results obtained showed that digestion efficiency was increased by lowering the flow rate while an efficient digestion was achieved in less than 10 min. proteinase K; microfluidic; magnetic beads; enzyme reactor

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