Kloniranje, ekspresija i karakterizacija fosforiboziltransferaza iz bakterije Escherichia coli

master's thesis

• Title (english): Cloning, expression and characterization of phosphoribosyltransferases in Escherichia coli
• Author: Tin Maršić
• Mentor: Christoph Loderer (mentor)
• Mentor: Nenad Malenica (sumentor)
• Committee member: Višnja Besendorfer (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Committee member: Inga Marijanović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019-09-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Nucleotides are an essential class of biological compounds necessary for proper function, growth and replication of all living organisms.
Alternative nucleotides have an application in medicine as anticancer and antiviral drugs. Newly emerging field of synthetic biology uses alternative nucleotides as the building blocks of xenonucleic acids (XNA)
Phosphoribosyltransferases (PRTs) are a group of enzymes utilized by living systems to synthetise nucleotides via salvage pathway. The shared property by these enzymes is the addition of nucleobases to ribose, which is previously activated in the form of 5-phosphoribosyl 1-pyrophosphate (PRPP).
The purpose of this work was to clone, express and purify five E. coli phosphoribosyltransferases and determine their optimal expression conditions, establish a purification protocol, working temperature and optimal pH as well as supstrate specificity. This work is a first step proof of the concept as part of a wider effort to produce a biosynthetic ribonucleotide synthesis pathway that would potentially allow for the production of alternative nucleotides for use in scientific research and biotechnology.
Five E.coli phosphoribosyltransferases were cloned, expressed at 37°C overnight, purified by His-Tag affinity and size-exclusion chromatography. Optimal buffer for all enzymes was established to be HEPES, and pH optima for HPRT and UPRT are 7.5 and 8 respectively, while optimal temperatures are 60° and 50°C respectively; 6-mercaptopurine was found to be taken up by XPRT and HPRT but not by APRT, while 5-fluorouracil is processed by UPRT but not by OPRT

Abstract (croatian)

Nukleotidi su jedna od temeljnih klasa bioloških spojeva potrebnih za ispravnu funkciju, rast i replikaciju svih živih organizama.
Alternativni nukleotidi nalaze primjenu u medicini kao citostatici i antivirotici. Novonastajuće polje sintetičke biologije koristi alternativne nuleotide kao gradivne jedinice ksenonukleinskih kiselina (XNA).
Fosforiboziltransferaze (PRT) su skupina enzima koja sintetizira nukleotide u procesu zvanom salvage pathway. Zajedničko svojstvo ovih enzima je dodavanje dušičnih baza na ribozu, koja je prethodno aktivirana u oblik 5-fosforibozil 1-pirofosfat (PRPP).
Svrha ovoga rada bila je klonirati, eksprimirati i pročistiti pet fosforiboziltransferaza iz E. coli te utvrditi optimalne uvjete ekspresije, protokol za pročišćavanje, radnu temperaturu te optimalni pH kao i specifičnost za suptrate. Ovaj rad je zamišljen kao prvi korak u nastojanju da se proizvede umjetni biosintetski put ribonukleotida koji bi omogućio sintezu alternativnih nukleotida za upotrebu u znanstvene ili biotehnološke svrhe.
Pet fosforiboziltransferaza iz E.coli je klonirano, eksprimirano preko noći pri 37°C, pročišćeno putem His-Tag afinitetne kromatografije te dodatno putem gel filtracije. Kao optimalni radni pufer za sve enzime, pokazao se HEPES. pH pri kojem su enzimi HPRT i UPRT najaktivniji su 7.5 odnosno 8, a optimalne temperature su 60°C odnosno 50°C. Pokazalo se da su XPRT i HPRT sposobni procesuirati 6-merkaptopurin, međutim to nije bio slučaj sa APRT-om. Također, 5-fluorouracil se pokazao supstratom za UPRT, ali ne i za OPRT.

• Keywords: E. coli; nucleotides; alternative nucleotides; phosphoribozyltransferase; characterization
• Keywords (croatian): E. coli; nukleotidi; alternativni nukleotidi; fosforiboziltransferaza; karakterizacija
• Language: english
• URN:NBN: urn:nbn:hr:217:694712
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2020-09-25
• Created on: 2020-06-09 10:56:02