Uporaba kalmodulina za renaturacijo rekombinantnega encimsko neaktivnega mutanta amoditoksina A

diplomsko delo

Matevž Merljak (Author), Igor Križaj (Mentor)

Abstract

Amoditoksini (Atx) so sekretorne fosfolipaze tipa A2 (sPLA2) iz strupa modrasa (Vipera ammodytes ammodytes), ki imajo presinaptično nevrotoksično aktivnost, imenovano tudi β-nevrotoksičnost. Navkljub mnogim odkritjem v zadnjih letih mehanizem njihovega delovanja še ni v celoti poznan, prav tako ni pojasnjena vloga encimske aktivnosti Atx v tem procesu. Za namen študija le-te so raziskovalci razvili rekombinantni mutant AtxA(D49S), ki nima encimske aktivnosti. Omenjen mutant pridobivamo iz bakterij, kjer se odlaga v obliki inkluzijskih telesc, zato ga je potrebno zviti v nativno obliko (renaturacija) ter očistiti. Ker je izkoristek tega procesa običajno precej nizek, je bil cilj pričujočega dela izboljšati izkoristek renaturacije z uporabo kalmodulina (CaM), poznanega liganda Atx, v vlogi šaperona. S takšnim postopkom smo uspešno izolirali nativno zvit mutant AtxA(D49S), vendar je dobljeni izkoristek nižji od pričakovanega.

Keywords

amoditoksin;nevrotoksičnost;rekombinantni proteini;renaturacija;kalmodulin;diplomska dela;

Data

Language:	Slovenian
Year of publishing:	2024
Typology:	2.11 - Undergraduate Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[M. Merljak]
UDC:	577.112(043.2)
COBISS:	201142275
Views:	41
Downloads:	2
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Refolding a recombinant enzymatically inactive mutant ammoditoxin A using calmodulin
Secondary abstract:	Ammoditoxins (Atx) are type A2 secretory phospholipases (sPLA2) from the venom of the nose-horned viper (Vipera ammodytes ammodytes) with presynaptic neurotoxicity (β neurotoxicity). Despite many recent discoveries in this field, the underlying mechanism and the role of Atx's enzymatic activity therein remains poorly understood. To gain better insight, researchers have developed an enzymatically inactive recombinant mutant AtxA(D49S). Mutant variant is produced in bacteria, where it aggregates into insoluble inclusion bodies. Thus, it needs to be refolded and purified before use, but the yield of such refolding is usually quite low. The goal of our work was to improve the refolding yield using a known Atx ligand calmodulin (CaM) as a chaperone. We show that AtxA(D49S) can be successfully refolded using such an approach, however the resulting yield is lower than expected.
Secondary keywords:	ammoditoxin;calmodulin;refolding;Beljakovine;Fosfolipaze;Univerzitetna in visokošolska dela;
Type (COBISS):	Bachelor thesis/paper
Study programme:	1000371
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages:	1 spletni vir (1 datoteka PDF (V, 39 str.))
ID:	24511964