|
|
Preparation of proteases for protein structure studies.
Jirečková, Barbora ; Man, Petr (advisor) ; Vaňková, Pavla (referee)
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is a method allowing the study of protein structure and dynamics. Its spatial resolution is given by the proteolysis step that is included in the HDX-MS workflow. Most widely used pepsin has however some limitations and use of a single protease often does not provide optimal spatial resolution. Several publications have emphasized the importance of the alternative proteases nepenthesin-2 (Nep-2) and aspergillopepsin (XIII) cleaving, in contrast to pepsin, after basic amino acids. In studies targeting proline-rich proteins, another enzyme, prolyl endoprotease from Aspergillus niger (AnPEP), is gaining importance. This work focuses on the characterization of immobilized AnPEP in combination with pepsin, aspergillopepsin or Nep-2 for their application in HDX-MS. First, columns with only one protease were tested on a set of model proteins. It was found that immobilized AnPEP did not have optimal cleavage characteristics compared to the other proteases. In order to combine the advantages of the proteases mentioned above, the model proteins were digested using columns with AnPEP coimmobilized with pepsin, Nep-2 or XIII and also using two protease columns in series (always AnPEP column with pepsin, Nep-2 or XIII column in both...
|
|
|
Characterization and application of proline-selective proteases.
Portašiková, Jasmína Mária ; Man, Petr (advisor) ; Kádek, Alan (referee)
Peptide bond formed by proline is resistant to most known proteases, therefore the discovery and isolation of proteases cleaving after this amino acid opens up new possibilities not only for protein characterization but also for industrial, food or pharmaceutical aplications. Aspergillus niger prolyl endopeptidase (AnPEP) is a proline-selective protease that is commercially available in a variety of products. In this bachelor thesis cleavage preferences of proteases AnPEP (Clarity Ferm) and ProAlanase (Promega) were characterized and compared. The effect of different conditions on the specificity of proteases, while cleavaging the mixture of model proteins was examined within this work. AnPEP cleavage preferences were characterized on complex protein mixtures and its immobilized form was tested as well. [IN CZECH] Keywords: ProAlanase, AnPEP, H/D exchange, structural proteomics, imobilized proteases [IN CZECH]
|
guest ::
