Priprava ter karakterizacija metaloproteaze Mpl iz patogene bakterije Listera monocytogenes

Korošec, Sara (author), Podobnik, Marjetka (mentor), Pavšič, Miha (co-mentor)

Bakterija Listeria monocytogenes je znotrajcelični patogen, ki prehaja med celicami in se deli v citosolu gostiteljske celice. Znotrajcelični cikel zajema pobeg iz vakuole, pri čemer sodeluje več virulentnih faktorjev, med drugim metaloproteaza Mpl. Gre za slabo raziskan bakterijski protein, zato smo se odločili za izolacijo in karakterizacijo. Pripravili smo več različnih konstruktov, ki so se med seboj razlikovali po afinitetni oznaki, prisotnosti signalnega zaporedja in pro- ali zreli obliki Mpl. Začetni pogoji gojenja bakterijskih celic in izražanja proteina so pokazali, da se rekombinanten proMpl izraža, a se nahaja v netopni frakciji, medtem ko izražanja zrele oblike Mpl nismo opazili. Cilj je postal pridobiti topno obliko proMpl. K temu izzivu smo pristopali sistematsko, tako da smo spreminjali oziroma vpeljevali po eno do dve spremenljivki hkrati. Preverili smo vpliv: i) temperature in časa izražanja, ii) liznega pufra, iii) metode bakterijske lize, a nobena od naštetih spremenljivk ni povečala topnosti proteina. Analiza z NaDS-PAGE je pokazala še prisotnost neznanega proteina velikosti približno 40 kDa, ki je predvidoma bakterijskega izvora. Delo smo nadaljevali s pripravo konstrukta, ki je omogočal izražanje proMpl v periplazemski prostor bakterije, vendar takšna sprememba ni povečala topnosti. Naslednji poskus je slonel na predpostavki, da ima proMpl težnjo k agregaciji v primeru prevelike količine izražanja. S tem namenom smo opazovali vpliv koncentracije induktorja in vrste bakterijskega gojišča, kar prav tako ni bila rešitev. Naslovili smo tudi možnost, da se proMpl ne uspe zviti do nativne konformacije. Pripravili smo konstrukt, ki omogoča izražanje nativnega proMpl brez dodatnih aminokislinskih ostankov. Novo pripravljen konstrukt smo izražali v prisotnosti in odsotnosti cinka dodanega v bakterijsko gojišče. Cinkov atom se nahaja v aktivnem mestu proMpl, prav zato bi lahko imel vlogo pri pravilnem zvitju. Poskusili smo še z izolacijo proMpl iz periplazemskega prostora, pri čemer celic nismo lizirali, ampak izvedli metodo modificiranega osmotskega šoka, vendar to ni povečalo topnosti Mpl. Pokazali smo, da rekombinantno izražanje proMpl zahteva posebne pogoje, ki jih kljub sistematskemu iskanju nismo odkrili. V modelu tridimenzionalne strukture, napovedane z AlphaFold2, lahko opazimo nestrukturirane dele in sklepamo na pomembnost propeptida. Predpostavljamo, da bi s sistematično pripravo različnih konstruktov proMpl na osnovi AlphaFold2 modela lahko pripravili take, ki bi se uspešno izražali v E.coli.

• Language: Slovenian
• Keywords: metaloproteaza, Listeria monocytogenes, Mpl, pro-encim, topnost
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-139849
• COBISS.SI-ID: 127862275
• Publication date in RUL: 08.09.2022

Secondary language

• Language: English
• Title: Preparation and characterisation of Mpl metalloprotease from the pathogenic bacterium Listeria monocytogenes

Abstract

Bacterium Listeria monocytogenes is an intracellular pathogen with the ability to spread between cells and divide in the cytosol of host cells. During intracellular cycle it becomes entrapped inside the vacuole, from which it escapes using virulence factors, including metalloprotease Mpl. Due to lack of information for this protein, we decided to isolate and characterize it. We prepared constructs which differed in affinity tag, signal peptide, sequence – either pro or mature form of Mpl. Cultivation of bacterium and expression of Mpl under initial conditions, showed that proMpl is expressed but remains in insoluble fraction, but mature Mpl is not even expressed. The goal became to obtain a soluble form of proMpl. We approached this challenge systematically, changing or introducing one or two variables at a time. Firstly, we checked the influence of i) temperature and time of expression, ii) lysis buffer, iii) bacterial lysis method, but none of the listed variables increased protein solubility. Results from NaDS-PAGE showed the presence of an unknow protein the size of approximately 40 kDa, which is most probably a bacterial protein. We continued the work by preparing a construct that allows expression of proMpl in the periplasmic space of the bacteria but did not see an increase in the solubility. The next assumption was that when proMpl is overexpressed it has a higher tendency to aggregate. We tested the influence of inducer concentration and type of bacterial medium, which was also not a solution. Next, we assumed that proMpl is not able to reach its native conformation, thus we prepared a construct which enables expression of proMpl without additional amino acid residues. We expressed proMpl from this newly prepared construct in the presence and absence of external zinc added to the bacterial culture medium. proMpl contains one zinc atom in its active site, therefore it could have an impact on correct folding. We also tried to isolate proMpl from the periplasmic space using modified osmotic shock, but this method was not the solution. We show that the expression of recombinant proMpl requires special conditions, which we did not discover despite a systematic search. Lastly, we determined the model of three-dimensional structure using AlphaFold2. The model predicts some unstructured regions, and we can also observe the importance of the propeptide. We assume that by systematically preparing various proMpl constructs based on AlphaFold2 model, we could prepare those that would be successfully expressed in E. coli.

• Keywords: metalloprotease, Listeria monocytogenes, Mpl, proenzyme, solubility