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Protein-Detergent Micellar Solutions for the Crystallization of Membrane Proteins: Some General Approaches and Experiences with the Crystallization of Pigment-Protein Complexes from Purple Bacteria
Published in Hartmut Michel, Crystallization of Membrane Proteins, 1991
We tend to use chromatofocusing as final purification step. As the pH of elution depends on the distribution of amino acids on the hydrophilic surface of the protein-detergent micelle, we consider this method to have the capacity of separating partially denatured material of the desired protein as well as impurities. Chromatofocusing separates proteins according to their isoelectric points, as does isoelectric focusing which has been shown to be a beneficial final purification step before crystallization30; but chromatofocusing has the further advantage that it does not require the application of an electric field which may cause damage to the protein.
Structural determinants for subnanomolar inhibition of the secreted aspartic protease Sapp1p from Candida parapsilosis
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Jiří Dostál, Jiří Brynda, Lucie Vaňková, Syeda Rehana Zia, Iva Pichová, Olga Heidingsfeld, Martin Lepšík
Sapp1p, used in this study, was an authentic enzyme purified from C. parapsilosis culture supernatant, as described previously23,32. C. parapsilosis strain P69 from the mycological collection of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic was cultivated in the YCB-BSA medium consisting of 1.2% (w/v) Yeast Carbon Base (Difco), 0.2% (w/v) BSA and 15 mM sodium citrate, pH 4. The culture supernatant obtained after approximately 70 h of cultivation and the removal of the cells by centrifugation (5000 g, 15 min) was subjected to ion-exchange chromatography, followed by gel filtration. Finally, Sapp1p and Sapp2p proteases were separated using chromatofocusing. Enzyme-activity assays were performed using the fluorogenic substrate Dabcyl-Glu-His-Val-Lys-Leu-Val-Glu-EDANS, which is cleaved at the Leu-Val bond by Sapp1p33.