Izlučivanje heterologno eksprimiranih proteina u kvasca Pichia pastoris pomoću sekvencije α-MF

master's thesis

• Title (english): Heterologous protein secretion in yeast Pichia pastoris using α-MF sequence
• Author: Silvija Cvitić
• Mentor: Ivan-Krešimir Svetec (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2009-12-14, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Metilotrofni kvasac Pichia pastoris je izvrstan domaćin za ekspresiju heterolognih proteina. Lako se uzgaja na jednostavnoj hranjivoj podlozi, a uporaba promotora gena AOX1 nudi prednosti kontrolirane i učinkovite indukcije. Ovaj kvasac izlučuje malu količinu endogenih proteina, zbog čega heterologno eksprimiran i izlučen protein čini većinu proteina hranjive podloge, što olakšava proces njegovog pročišćavanja. Ipak, većina visokomolekularnih proteina se ne izlučuje u podlogu već ostaje u periplazmi stanice. Kako bi se kvasac P. pastoris ustanovio kao učinkovit domaćin za ekspresiju ovih proteina, nužno je poboljšanje njegovog sekretornog kapaciteta. U ovom radu opisan je pokušaj izlučivanja tri modelna proteina fuzijom sa signalnom sekvencijom α-MF iz kvasca Saccharomyces cerevisiae. Osim toga, istražen je i utjecaj razine ekspresije nekih gena koji sudjeluju u izgradnji stanične stijenke na sekretorni kapacitet kvasca P. pastoris.

Abstract (english)

The methylotrophic yeast Pichia pastoris is an outstanding host for heterologous expression. It grows on relatively simple media and the AOX1 promoter offers an efficient and easily controlled induction of gene expression. Since this yeast secrets only low levels of endogenous proteins, heterologously expressed and secreted protein comprises the vast majority of total protein in the growth medium simplifying purification of the heterologous protein. However, the proteins of high molecular weight usually remain the periplasm of the yeast cell. To establish P. pastoris as an efficient expression host for such proteins, an improvement of its secretory capacity would be necessary. This work reports an attempt to secret three model proteins by fusion to α-MF sequence originated from Saccharomyces cerevisiae. Additionally, it was investigated if level of particular proteins involved in the cell wall synthesis affects the efficiency of protein secretion in the yeast P. pastoris.

• Keywords: izlučivanje heterolognih proteina; kvasac P. pastoris; sekvencija α-MF; stanična stijenka
• Keywords (english): heterologous protein secretion; yeast P. pastoris; α-MF sequence; cell wall
• Language: croatian
• URN:NBN: urn:nbn:hr:159:572459
• Study programme: Title: Biotechnology; specializations in: Biochemical-Microbiology; Course: Biochemical-Microbiology; Study programme type: university; Study level: graduate; Academic / professional title: diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki (diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-09-01 10:27:15