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Optimizacija biosinteznega postopka za pridobitev in izolacijo rekombinatnega pernizina z bakterijo Streptomyces rimosus
Stare, Nastja (Author), Petković, Hrvoje (Mentor) More about this mentor... This link opens in a new window, Kranjc, Luka (Co-mentor)

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Abstract
Pernizin je ekstracelularna proteaza arheje Aeropyrum pernix, ki spada v skupino subtilizinu podobnih proteaz. Nativni pernizin ima sposobnost razgradnje prionov, ki se pojavljajo pri Creutzfeldt – Jakobovi bolezni, vendar je njegovo pridobivanje s pomočjo arheje A. pernix zaradi nizkih donosov in zahtevnih pogojev rasti neekonomično. V okviru raziskovalnega dela magistrske naloge, smo kot gostiteljski sistem za heterologno izražanje pernizina uporabili bakterijo Streptomyces rimosus v katerega smo transformirali replikativni E. coli – Streptomyces prenosljivi plazmid pVF, ki je vseboval nukleotidni zapis za rekombinantni pernizin. Tekom eksperimentalnega dela smo transformirali genski zapis za produkcijo pernizina v bakterijo Streptomyces rimosus ∆OTC, uspešnost izražanja rekombinantnega pernizina pa preverili z uporabo afinitetne kromatografije in vizualizacijo na poliakrilamidnem gelu ter dokazali njegovo proteolitično aktivnost z azokazeinskim testom in cimografijo. Rezultati eksperimentov so pokazali, da je bakterija Streptomyces rimosus primerna za produkcijo pernizina s heterologno ekspresijo, nadaljne raziskave pa bodo pokazale, če je rekombinatni pernizin enako odporen na denaturante in če je zmožen razgradnje infektivnih prionov.

Language:Slovenian
Keywords:Streptomyces rimosus, serinske proteaze, pernizin, heterologno izražanje, proteolitična aktivnost, plazmid, afinitetna kromatografija
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2019
Publisher:[N. Stare]
UDC:602.3+602.4:579.873.7:577.15
COBISS.SI-ID:5041016 Link is opened in a new window
Views:66
Downloads:37
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Secondary language

Language:English
Title:Optimisation of biosynthesis procedure for acquisition and isolation of recombinant pernisine with bacteria Streptomyces rimosus
Abstract:
Pernisine in an extracellular protease, isolated from archaeon Aeropyrum pernix, which belongs to the group of subtilisin-like proteases. Native persine has the ability to degrade prions which cause Creutzfeldt – Jakob disease. The production of native pernisine from the archea is not economical, mainly because of low yields and demanding growth conditions, which can not be ensured by the regular indutrial scale fermenters. As a part of this research project, we used bacterium Streptomyces rimosus as a host for heterologous expression of the recombinant pernisine. S. rimosus ∆OTC was transformed with a replicative shuttle E. coli – Streptomyces plasmid construct pVF, which contained nucleotide sequence encoding pernisine. The aim of the Master's thesis was to successfully transform the plasmid for the production of recombinant pernisine into S. rimosus. The successful production of pernisine was confirmed by partial isolation of pernisine with affinity chromatography and further analysis with SDS – PAGE. Proteolytic activity of recombinant pernisine was measured with the use of asocasein assay and zymography. We have confirmed successful production of pernisine by the selected transformants of S. rimosus. We have thus confirmed that S. rimosus is suitable host for production of recombinant pernisine. Further evaluation of the properties of pernisine produced this way will be necessary to establish the capability of the recombinant pernisine to degrade infectious prion proteins.

Keywords:Streptomyces rimosus, serine protease, pernisine, heterologous expression, proteolytic activity, plasmid, affinity chromatography

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