Priprava rekombinantnih bakterij vrste Escherichia coli za izražanje ciljnega proteina in prenos proizvodnje v večje merilo.

Tumpej, Tadeja (author), Podgornik, Aleš (mentor)

Bioprocesništvo je bistveni del prehrambne, kemijske in farmacevtske industrije. Pri bioprocesih uporabljamo mikroorganizme, živalske, rastlinske in sesalske celice za produkcijo novih produktov. Rekombinantni proteini predstavljajo pomemben del farmacevtskih in biotehnoloških produktov. Kadar je le mogoče, jih izražamo v mikroorganizmih, ki imajo visoko specifično hitrost rasti ter visok doprinos, kar se odraža v visoki produktivnost. Glavni cilj magistrskega dela je bil razvoj učinkovitega procesa za pridobivanje rekombinantnega proteina v večjem merilu. Potrebno je bilo celovito obravnavanje procesa, saj le-to omogoča učinkovit razvoj proizvodnega procesa. Bioproces smo najprej okarakterizirali v Erlenmeyerjevih steklenicah na stresalniku. V Erlenmeyerjevih steklenicah smo določili volumetrični koeficient prenosa kisika (kLa), spremljali kinetiko rasti mikroorganizma Escherichia coli in produkcijo rekombinantnih proteinov. Razvili smo zaključne procese za izolacijo rekombinantnega proteina. S povečevalnimi metodami, ki so temeljile na konstantnem kLa in ostalih pogojih gojenja (gojišče, temperatura, vrsta bioprocesa), smo prenesli produkcijo proteinov v mešalni bioreaktor. Nismo dosegli enake kinetike rasti in produktivnosti procesa v obeh sistemih (Erlenmeyerjeva steklenica in mešalni bioreaktor). Dosegli smo podoben trend produkcije rekombinantnega proteina in produktivnost celic.

• Language: Slovenian
• Keywords: bioproces, produkcija rekombinantnih proteinov, Escherichia coli, volumetrični koeficient prenosa kisika, povečevalne metode, mešalni bioreaktor, zaključni procesi, imobilizirana kovinska afinitetna kromatografija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [T. Tumpej]
• Year: 2017
• PID: 20.500.12556/RUL-98159
• UDC: 602.3/.4:579.84:604.4(043.2)
• COBISS.SI-ID: 8866937
• Publication date in RUL: 19.11.2017

TUMPEJ, Tadeja, 2017, Priprava rekombinantnih bakterij vrste Escherichia coli za izražanje ciljnega proteina in prenos proizvodnje v večje merilo. [online]. Master’s thesis. T. Tumpej. [Accessed 18 May 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=98159

Secondary language

• Language: English
• Title: Preparation of the recombinant bacteria Escherichia coli for expression of the target protein, and scale-up of fermentation processes

Abstract

Bioprocessing is an essential part of many food, chemical, and pharmaceutical industries. Bioprocesses include the use of microorganisms and animal, plant mammalian cells for the production of new products. Recombinant proteins today represent an important part of pharmaceutical and biotechnological products. Whenever possible, they are expressed in microorganisms that have a high specific growth rate and high contribution, which is reflected in high productivity. The main task of the master's thesis is the development of recombinant protein production on a larger scale. The whole production process has to be characterized to enable efficient production process development. First we characterized bioprocesses in the shake flask. We determined the volumetric coefficient of oxygen transfer (kLa), the bacterial growth curve and the expression of recombinant proteins in the shake flask. We developed the processes for the isolation of the recombinant protein. With scale-up methods based on constant kLa and constant conditions of cultivation (medium, temperature, bioprocess type), we transferred the production of recombinant proteins to the stirred tank bioreactor. We did not reach the same growth kinetics and productivity at both scales. We achieved the similar trend of expression of recombinant protein and a similar cell productivity.

• Keywords: bioprocess, production of the recombinant protein, Escherichia coli, scale-up, volumetric oxygen transfer coefficient, stirred tank bioreactor, downstream processing, immobilized metal affinity chromatography