Prekomjerna ekspresija i biokemijska karakterizacija mutanata humane dipeptidil-peptidaze III iz zloćudnih tumora

master's thesis

• Title (english): Overexpression and biochemical characterization of human dipeptidyl-peptidase III mutants found in cancer
• Author: Goran Pipalović
• Mentor: Mihaela Matovina (mentor)
• Committee member: Aleksandra Maršavelski (predsjednik povjerenstva)
• Committee member: Željka Soldin (član povjerenstva)
• Committee member: Vesna Petrović Peroković (član povjerenstva)
• Committee member: Morana Dulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2018-09-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Dipeptidil-peptidaze III (DPP III) su metalopeptidaze iz porodice M49 koje hidroliziraju
dipeptide s N-kraja peptidnih supstrata veličine 3 do 10 aminokiselina. Fiziološka funkcija
ove peptidaze nije još do kraja razjašnjena, ali pretpostavlja se da ljudska DPP III osim uloge
u završnim koracima razgradnje peptida u stanici ima ulogu i u nekoliko patofizioloških
procesa, uključujući razvitak raka. U cBioPortal bazi podataka nalaze se sekvencirani uzorci
DNA izolirane iz ljudskih tumora te su nađene mutacije u kodirajućoj regiji gena za humanu
DPP III. Odabrane su tri mutacije kako bi se ispitao učinak istih na biokemijska svojstva i
aktivnost humane DPP III.
Istraživanje se provodilo na mutantima humane DPP III: E451K, R638L i R638W koji su
dobiveni ciljanom in vitro mutagenezom ekspresijskih plazmida. Nakon tog slijedila je
ekspresija mutanata u bakteriji E. coli te njihova izolacija i pročišćavanje afinitetnom
kromatografijom na Ni-NTA koloni. Mutanti su podvrgnuti raznim testovima aktivnosti i
mjerenju kinetike te su analizirani nativnom elektroforezom i izoelektričnim fokusiranjem na
gelu. Dobiveni parametri za mutante uspoređeni su s onima „divljeg“ tipa.

Abstract (english)

Dipeptidyl peptidases III (DPP III) are members of the M49 metalopeptidase family which
hydrolyze dipeptides from the N-termini of 3 to 10 aminoacids long peptides. Their
physiological function is not yet fully understood, but it is assumed that the human DPP III,
apart from the role in the final steps of peptide degradation in the cell, has the role in several
pathophysiological processes, including the development of cancer. The cBioPortal database
contains sequenced DNA samples isolated from human cancers and mutations in the coding
region of the human DPP III gene are found. Three mutations were selected to examine their
effect on the biochemical properties and the activity of human DPP III.
The study was performed on human DPP III: E451K, R638L and R638W mutants obtained
by site-directed in vitro mutagenesis of expression plasmids, followed by the expression of
mutants in E. coli, and the isolation and purification by affinity chromatography on Ni-NTA
column. The mutants were subjected to various activity assays, kinetics measurement, and
they were also analysed by native and isoelectric focusing gel electrophoresis. The obtained
mutant parameters were compared to those of the wild type.

• Keywords: Dipeptidil-peptidaza III; kinetika; mutageneza; mutanti; razgradnja proteina; tumori
• Keywords (english): Degradation of proteins; dipeptidyl-peptidase III; kinetics; mutagenesis; mutants; tumor
• Language: croatian
• URN:NBN: urn:nbn:hr:217:896663
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2020-09-27
• Created on: 2018-11-23 11:47:47