Razvoj presejalnega sistema za detekcijo homodimerizacije proteinov v bakteriji Escherichia coli

magistrsko delo

Liza Ulčakar (Avtor), Marko Novinec (Mentor), Marko Dolinar (Član komisije za zagovor), Gregor Gunčar (Član komisije za zagovor)

Povzetek

Ključne besede

oligomerizacija proteinov;homodimerizacija;proteinski inženiring;papainu podobne cisteinske proteaze;dipeptidil-peptidaza I;ToxR;LexA;magistrska dela;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2022
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[L. Ulčakar]
UDK:	577.112(043.2)
COBISS:	134085635
Št. ogledov:	286
Št. prenosov:	2
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	Development of a screening assay for detection of protein homodimerization in Escherichia coli
Sekundarni povzetek:	Oligomerization is an important aspect of protein structure, as it can improve some existing protein properties and even lead to previously nonexistent ones. The development of artificial oligomers is therefore an exciting area of protein engineering. Papain-like cysteine proteases are among the most used industrial enzymes and would potentially benefit from oligomeric structure. The goal of our research group is to produce dimeric forms of these proteins using a semi-rational approach. The aim of this master thesis was to develop a screening assay for the identification of homodimeric variants from a library of mutant protein variants. We have prepared three systems for the detection of homodimerization, with mechanisms of action based on the activity of chimeric proteins, constructed on the basis of transcription factors LexA from E. coli, ToxR from V. cholerae and the cytosolic form of ToxR, respectively. The chimeras were prepared by replacing the coding sequences for the dimerization domains of LexA and ToxR with those of the studied protein. The fusion partners in the chimeras were connected by a flexible linker. If the chimera homodimerized, it would form an active transcription factor that would activate transcription of a reporter protein in the case of ToxR or repress transcription in the case of LexA. To characterize these systems, we prepared controls that contained a protein with a known KD value of dimerization as a fusion partner. First, we prepared systems that used green fluorescent protein (GFP) as the reporter and named them LEXGFP, TOXGFP and cTOXGFP (i.e. the cytosolic form of TOXGFP), respectively. We characterized these systems by measuring the fluorescence of GFP in cells transformed with control constructs. Of the three systems, only TOXGFP worked as intended. Unfortunately, using GFP as the reporter is not very convenient for screening purposes. Therefore, we prepared new variants of the TOXGFP system by replacing the reporter. The TOXKAN variant used aminoglycoside 3'-phosphotransferase as the reporter, which should allow screening with positive selection in medium containing kanamycin. The TOXBLUE variant, on the other hand, used the α-fragment of β-galactosidase as the reporter, which should enable blue-white screening in combination with an appropriate strain of E. coli. We tested these variants with the control constructs and TOXBLUE proved not to be sensitive enough for use in screening. In contrast, TOXKAN proved to be suitable for screening, but only at high concentrations of kanamycin. Further research is needed to modify the sensitivity of these systems for use in homodimerization assays. Finally, we attempted to determine the oligomeric state of prodipeptidyl-peptidase I in E. coli, using TOXGFP and TOXKAN. We came to the conclusion that the protease forms homodimers. However, these findings should be investigated further.
Sekundarne ključne besede:	homodimerization;ToXR;LexA;dipeptidyl-peptidase I;Beljakovine;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS):	Magistrsko delo/naloga
Študijski program:	1000377
Konec prepovedi (OpenAIRE):	2024-10-06
Komentar na gradivo:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Strani:	81 str.
ID:	16671186