diplomsko delo
Tinkara Božič (Author), Marina Klemenčič (Mentor)

Abstract

Cisteinska endopeptidaza CEP2 iz zelene alge Chlamydomonas reinhardtii sodi v družino papainu podobnih cisteinskih proteaz (angl. papain like cysteine proteases, PLCP). Predstavniki družine PLCP so v rastlinah povezani z regulirano celično smrtjo, ki ji v fizioloških pogojih (npr. razvoj rastline) lahko rečemo tudi programirana celična smrt (angl. programmed cell death, PCD). Med PLCP so pomembne proteaze RD21 iz modelne rastline Arabidopsis thaliana, ki sodelujejo pri sprožitvi PCD pri rastlinah in njihovemu imunskemu odgovoru proti patogenom. Cisteinska endopeptidaza CEP2 iz C. reinhardtii je sorodna predstavnikom iz poddružine RD21B, zato ima morda podobno vlogo v rastlinski celični smrti. Da bi lažje predvideli biokemijske lastnosti CEP2, smo proteazo najprej bioinformacijsko analizirali. Na podlagi aminokislinskega zaporedja proteina CEP2 smo s spletnimi orodji pripravili model njegove terciarne strukture. Aminokislinsko zaporedje CEP2 smo tudi primerjali z zaporedjem RD21B in iz poravnave obeh predvideli vlogo posameznih delov zaporedja in obseg določenih domen v polipeptidni verigi. Laboratorijsko delo smo začeli s pripravo rekombinantnega proteina CEP2 v bakterijskih celicah Escherichia coli, seva Rosetta-gami 2. Temu je sledila izolacija proteina, preučevali pa smo tudi aktivacijo proteina CEP2 zaradi znižanja pH. Opazili smo, da se protein delno procesira že med izražanjem in izolacijo, saj se mu odcepi C-končna domena, ter da je očiščen protein približno enako aktiven tako brez predhodne inkubacije v pufru z znižanim pH kot po njej. Poleg tega smo pripravili tudi pH-profil aktivnosti encima in pokazali, da je CEP2 optimalno aktiven v rahlo kislem območju, pri pH 5,0. Na podlagi rezultatov poskusa, s katerim smo zavrli avtokatalitično delovanje encima, sklepamo, da se protein CEP2 ne avtoprocesira, ampak je za cepitev proteina zaslužna neka proteaza, ki je prisotna v lizatu E. coli, a še ni identificirana.

Keywords

Chlamydomonas reinhardtii;papainu podobne cisteinske proteaze;rastlinska celična smrt;CEP2;izražanje in čiščenje proteinov;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [T. Božič]
UDC: 577.15(043.2)
COBISS: 117152771 Link will open in a new window
Views: 186
Downloads: 125
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Other data

Secondary language: English
Secondary title: Expression of recombinant CEP2 protease from the green alga Chlamydomonas reinhardtii and its activation in Escherichia coli
Secondary abstract: Cysteine endopeptidase CEP2 from the green alga Chlamydomonas reinhardtii belongs to papain-like cysteine proteases (PLCPs). In plants, PLCPs are associated with regulated cell death, also called programmed cell death (PCD) under physiological conditions (such as plant development). A subfamily of PLCPs from the model plant Arabidopsis thaliana, RD21, are particularly important enzymes involved in PCD, as they initiate PCD and mediate the plant-pathogen immune response. Cysteine endopeptidase CEP2 from C. reinhardtii shares many similarities with members of the RD21B subfamily of enzymes, which leads us to believe it may have a similar role in plant cell death as RD21B enzymes. Firstly, in order to more easily predict biochemical properties of CEP2, we preformed a bioinformatic analysis of the protease. Based on its amino acid sequence and the use of an internet tool we prepared a model tertiary structure of the protein. We also compared the sequences of CEP2 and RD21B, respectively, and used that knowledge to predict the role and position of specific parts of the sequence and the domains in the polypeptide chain. Our laboratory work began with the preparation of the recombinant protein CEP2 in Escherichia coli strain Rosetta-gami 2. This was followed by protein isolation and subsequent analysis of the protein's activation in the presence of a mildly acidic buffer. We noticed the protein gets processed during expression and isolation and that it is similarly active with and without incubation in a buffer with a lower pH value. Subsequenly, we determined the protein's acitivity pH profile which helped us establish the pH value for the protein's optimal activity which is slighly acidic, at 5.0. Lastly, based on results from an experiment during which the protein's autocatalytic activity was inhibited, we predict that the activation happens by means of another protease rather than by autoprocessing. The identitiy of said protease, which is present in the E. coli lysate, remains to be elucidated.
Secondary keywords: Chlamydomonas reinhardtii;plant cell death;papain-like cysteine proteases;CEP2;protein expression and purification;Encimi;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 35 str.
ID: 15857866