magistrsko delo
Mladen Davidovikj (Author), Marko Novinec (Mentor), Miha Pavšič (Thesis defence commission member), Aljaž Gaber (Thesis defence commission member)

Abstract

Katepsin B je cisteinska peptidaza, ki sodi v skupino papainu podobnih peptidaz. Katepsin B je monomeren in globularen encim, ki je prisoten v večini živih organizmov ter sodeluje pri raznih osnovnih fizioloških in patoloških procesih. V tej raziskavi smo poskusili pripraviti dimerno različico katepsina B. Oligomerizacija encimov v določenih primerih lahko povzroči spremembe v njihovih lastnostih, kot so povečana stabilnost ali večja katalitična učinkovitost. Zato smo poskusili ta proces posnemati v laboratorijskih poskusih. Najprej smo izbrali 19 mest v aminokislinski verigi na spodnji površini proteina glede na standardno orientacijo, na katerih smo pripravili knjižnice mutant z metodo mestna-specifična saturacijska mutageneza. Pripravljene knjižnice smo presejali na dimerizacijo z uporabo bakterijskega dvohibridnega sistema na osnovi adenilat ciklaze in tako dobili 15 morebitnih dimerov z mutacijami v šestih različnih mestih v aminokislinski verigi. Poskusili smo izraziti in izolirati štiri morebitne dimere, kar nam je uspelo le v enem primeru. Analizirali smo dimer, sestavljen iz variant catB_SNS154,156WNH in catB_SNS154,156GNP, s kromatografijo na osnovi ločevanja po velikosti. Ugotovili smo, da pri pogojih, pri katerih smo izvajali poskus, ni prišlo do dimerizacije. Različici smo aktivirali avtokatalitično. Encimsko aktivnost izoliranim različicam smo potrdili s kinetično metodo in določili njihovi konstanti K$_m$, ki sta imeli vrednosti 34±6 µM in 142±54 µM.

Keywords

oligomerizacija proteinov;dimeri;katepsin B;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [M. Davidovikj]
UDC: 577.15(043.2)
COBISS: 218988035 Link will open in a new window
Views: 81
Downloads: 25
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Other data

Secondary language: English
Secondary title: Preparation of dimeric variants of human cathepsin B using site-specific saturation mutagenesis
Secondary abstract: Cathepsin B is a cysteine peptidase belonging to the papain-like peptidase group. It is a monomeric and globular enzyme present in most living organisms, participating in various fundamental physiological and pathological processes. In this study, we attempted to prepare a dimeric version of cathepsin B. Oligomerization of enzymes can, in certain cases, lead to changes in their properties, such as increased stability or greater catalytic efficiency. Therefore, we attempted to mimic this process in laboratory experiments. Initially, we selected 19 positions in the amino acid chain on the lower surface of the protein, according to the standard orientation, to create mutant libraries using the site-specific saturation mutagenesis method. We screened the prepared libraries for dimerization using bacterial adenylate cyclase based two-hybrid system, identifying 15 potential dimers with mutations at 6 different positions in the amino acid chain. We attempted to express and isolate 4 potential dimers, succeeding in only one case. We analyzed a dimer composed of variants catB_SNS154,156WNH and catB_SNS154,156GNP using size-exclusion chromatography. We found that under the conditions in which the experiment was conducted, no dimerization occurred. We activated the variants autocatalytically. The enzyme activity of the isolated variants was confirmed using kinetic methods and we determined their K$_m$ values which were and 34±6 µM in 142±54 µM respectively.
Secondary keywords: cathepsin B;dimer;mutagenesis;Katepsini;Mutageneza;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 1 spletni vir (1 datoteka PDF ([63] str.))
ID: 25381218