magistrsko delo
Zina Ravnik (Avtor), Matej Butala (Recenzent), Hrvoje Petković (Mentor), Luka Kranjc (Komentor)

Povzetek

Bakterija Streptomyces rimosus, producent antibiotika oksitetraciklina (OTC) je industrijsko pomembna bakterija, ki ima GRAS (splošno priznan kot varen) status. Poleg OTC v gojišče učinkovito izloča tudi proteine, zato smo v obsegu magistrske naloge želeli na primeru termostabilne serinske proteaze camizina arheje A. camini ovrednotiti uporabnost bakterije S. rimosus za produkcijo heterolognih proteinov. Proteaza caminizin je glede na aminokislinsko zaporedje zelo podobna pernizinu iz arheje A. pernix, ki je bila nedavno sintetizirana v bakteriji S. rimosus. Konstruirali smo različne konstrukte plazmida pAB04, ki se integrirajo v genom S. rimosus in vsebujejo sekrecijsko sekvenco srt, histidinski repek, konstitutivni promotor ermE* in kodonsko optimizirane zapise za: zrel caminizin (pAB04 ermE* srT Per AC CO HT), procaminizin (pAB04 ermE* srT ProPer AC CO HT) ali preprocaminizin (pAB04 ermE* srT PreProPer AC CO HT). Po določitvi potencialno zanimivih klonov z azokazeinskim testom in cPCR (ang. colony PCR), smo preverili in potrdili sposobnost izločanja tarčnega proteina v gojišče. Uspešno smo detektirali vse tri oblike caminizina, ki smo jih nato identificirali na NaDS-PAGE, cimogramu in potrdili s prenosom po Westernu. Čeprav smo jasno potrdili prisotnost rekombinantnega caminizina, je bila dobljena koncentracija nizka, pojavljali so se tudi drugi nativni proteini. Kljub temu uspeli uspešno identificirati vse tri oblike caminizina. Sledilo je vrednotenje aktivnosti rekombinantnega caminizina. Iz dobljenih rezultatov lahko zaključimo, da je bakterija S. rimosus ΔOTC uporaben gostitelj za izražanje heterolognih proteinov, pri čemer je potrebno z dodatnim razvojem še intenzivno optimizirati gostiteljski sev S. rimosus in razviti boljši postopek izolacije proteina.

Ključne besede

streptomicete;Streptomyces rimosus;rekombinantni proteini;heterologno izražanje;caminizin arheje Aeropyrum camini;proteolitična aktivnost;afinitetna kromatografija;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL BF - Biotehniška fakulteta
Založnik: [Z. Ravnik]
UDK: 602.3:579.873.7:577.1:543.544
COBISS: 5180536 Povezava se bo odprla v novem oknu
Št. ogledov: 804
Št. prenosov: 345
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Expression of heterologous proteins in industrial host Streptomyces sp.
Sekundarni povzetek: Bacterium Streptomyces rimosus is used in industry for production of antibiotic oxytetracycline (OTC) and therefore has GRAS (Generally Recognized as Safe) status. A number of proteins are secreted in medium. Therefore, the bacterium Streptomyces rimosus was tested for production of heterologous proteins, where gene encoding thermostable serine protease caminisine originally isolated from A. camini was integrated into S. rimosus genome. Amino acid composition of caminisine is very similar to pernisine from A. pernix, which was recently produced in S. rimosus. We designed several plasmid construced based on integrative plasmid pAB04, which included srt signal sequence, histidine tag, strong constitutive promoter ermE* and codon optimized DNA sequences for: mature caminisine (pAB04 ermE* srT Per AC CO HT), procaminisine (pAB04 ermE* srT ProPer AC CO HT) and preprocaminisine (pAB04 ermE* srT PreProPer AC CO HT). Based on azocasein assay and cPCR (colony PCR), we selected potentially interesting clones secreting recombinant caminisine for isolation with affinity chromatography. By applying SDS-PAGE, zymography and Western-blot assays, we confirmed the presence of different versions of recombinant pernisine. The amount of recombinant enzyme isolated with affinity chromatography was low and in addition few native were observed on the SDS-PAGE gels in the elution fractions. The highest specific proteolitic activity among different versions of the recombinant caminisine was observed for fully processed recombinant caminisine produced by S. rimosus ΔOTC transformed with pAB04 ermE* srT Per AC CO HT. Finally, it can be concluded that S. rimosus is potentially useful host for production of caminisine, however additional optimization of host strain and protein isolation methods would be needed.
Sekundarne ključne besede: Streptomyces;Streptomyces rimosus;recombinant proteins;heterologous expression;caminisine originally isolated from A. camini;proteolytic activity;affinity chromatography;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 0
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Strani: XII, 72 f., [6] f. pril.
ID: 11491730