magistrsko delo
Povzetek
Proteaza kaminizin izolirana iz arheje Aeropyrum camini spada v skupino subtilizinu podobnih proteaz. Glede na aminokislinsko zaporedje je zelo podobna že karakterizirani proteazi pernizin iz arheje Aeropyrum pernix. Ta lahko razgrajuje prionske proteine. Zaradi nizkih donosov in ekstremnih pogojev rasti je pridobivanje proteaze s pomočjo arheje A. camini v večjem merilu praktično nemogoče. V okviru magistrskega dela smo kot gostiteljski organizem za heterologno izražanje fuzijskega kompleksa fluorescentnega proteina mCherry in kaminizina uporabili bakterijo Streptomyces rimosus. Predhodno konstruirane plazmidne vektorje za izražanje rekombinantnih proteinov kaminizin, pro-kaminizin, mCherry-kaminizin in mCherry-pro-kaminizin v bakteriji S. rimosus (pAB04_ermE*_srT_kaminizin / pro-kaminizin / mCherry-kaminizin / mCherry pro-kaminizin_AC CO HT) smo z elektroporacijo vnesli v modificiran gostiteljski sev bakterije S. rimosus ΔOTC, pri kateri je celotna genska skupina za biosintezo oksitetraciklina odstranjena. Transformante bakterije S. rimosus smo nato gojili v različnih gojiščih in primerjali produkcijo in zunajcelično aktivnost obeh proteinov v fuzijskem proteinskem konstruktu. Prisotnost proteina mCherry smo določali z merjenjem fluorescence, aktivnost različic kaminizina pa z azokazeinskim testom. Dokazali smo korelacijo med intenziteto fluorescence in aktivnostjo kaminizina. Uspešnost izražanja rekombinantnega kaminizina smo po izolaciji encima iz supernatanta z obarjanjem s TCA oz. z nikelj-afinitetno kromatografijo ovrednotili z NaDS-PAGE, proteolitično aktivnost pa potrdili s cimografijo in azokazeinskim testom. Z afinitetno kromatografijo nismo uspeli močno koncentrirati kaminizina iz supernatanta, kar se je odražalo v nizki intenziteti lis na NaDS-PAGE gelu. S pomočjo TCA obarjanja smo izolirane celokupne zunajcelične proteine vizualizirali na NaDS-PAGE gelu in na cimogramu, pri čemer smo preverili vpliv predhodne temperaturne aktivacije na vsebnost proteinov. Dokazali smo, da ima fuzijski protein mCherry-kaminizin enako proteinazno aktivnost kot sam kaminizin. Preverili smo tudi vpliv različnih temperatur, vrednosti pH in koncentracije NaDS na aktivnost kaminizina. Ugotovili smo, da je aktivnost kaminizina najvišja pri temperaturi 80 °C in pH 8,0.
Ključne besede
streptomicete;Streptomyces rimosus;heterologno izražanje;kaminizin;mCherry;fuzijski protein;fluorescenca;proteolitčna aktivnost;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2024 |
Tipologija: |
2.09 - Magistrsko delo |
Organizacija: |
UL BF - Biotehniška fakulteta |
Založnik: |
[T. Nusdorfer] |
UDK: |
606:604.4:577.15:602.3:579.873.7(043.2) |
COBISS: |
193999619
|
Št. ogledov: |
74 |
Št. prenosov: |
18 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
Evaluation of fusion reporter system of fluorescent protein mCherry and termostable protease caminisine in Streptomyces rimosus |
Sekundarni povzetek: |
The protease caminisine, isolated from the archaea Aeropyrum camini, belongs to the group of subtilisin-like proteases. Its amino acid sequence is similar to the previously characterised protease pernisine from the archaea Aeropyrum pernix, which can degrade prion proteins. Due to the low yields and extreme growth conditions of A. camini, large-scale production of the protease caminisine is not feasible. In the framework of the MSc thesis, the bacterium Streptomyces rimosus was used as a heterologous host for the production of an engineered caminisine complex with the fluorescent protein mCherry. Constructed plasmid vectors for the production of engineered proteins caminisine, pro- caminisine, mCherry-caminisine and mCherry-pro-caminisine in S. rimosus (pAB04_ermE*_srT_kaminizin / pro-kaminizin / mCherry-kaminizin / mCherry-pro-kaminizin_AC CO HT) were transformed by electroporation into S. rimosus ΔOTC, which has the gene cluster encoding oxytetracyline biosynthesis deleted. The transformants were then grown in different media and the extracellular activities of the constructed fusion proteins were compared. The presence of mCherry was determined by fluorescence measurements and the activity of caminisine variants was determined by an azo-casein assay. We demonstrated a correlation between fluorescence intensity and caminisine activity. After isolation of the enzyme from the supernatant by TCA precipitation or nickel-affinity chromatography, the expression efficiency of the recombinant caminisine was assessed by NaDS-PAGE and the proteolytic activity was confirmed by cimography and the azo-casein assay. Affinity chromatography failed to significantly concentrate the caminisine from the supernatant, which was reflected by the low intensity of the protein bands on the NaDS-PAGE gel. Total extracellular proteins isolated by TCA were visualised on NaDS-PAGE gels and on a cimogram to verify the effect of prior temperature activation on protein content. We demonstrated that the mCherry-caminisine fusion protein has the same proteinase activity as caminisine alone. The effect of different temperatures, pH values and NaDS concentrations on the activity of caminisine was also tested. We found that the highest activity of caminisine as a fusion protein is highest at 80 °C and pH 8,0. |
Sekundarne ključne besede: |
Streptomyces;Streptomyces rimosus;heterologous expression;caminisine;mCherry;fusion protein;fluorescence;proteolytic activity; |
Vrsta dela (COBISS): |
Magistrsko delo/naloga |
Študijski program: |
0 |
Konec prepovedi (OpenAIRE): |
1970-01-01 |
Komentar na gradivo: |
Univ. v Ljubljani, Biotehniška fak., Oddelek za agronomijo |
Strani: |
1 spletni vir (1 datoteka PDF (XI, 62 str., [3] str. pril.)) |
ID: |
23195773 |