Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori

Mišković, Marija Zora; Wojtyś, Marta; Winiewska-Szajewska, Maria; Wielgus-Kutrowska, Beata; Matković, Marija; Domazet Jurašin, Darija; Štefanić, Zoran; Bzowska, Agnieszka; Leščić Ašler, Ivana (2024) Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori. International Journal of Molecular Sciences, 25 (14). ISSN 1422-0067

PDF - Published Version - article Available under License Creative Commons Attribution. Download (5MB)

Abstract

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked.

Item Type:	Article
Uncontrolled Keywords:	adenylosuccinate synthetase; Helicobacter pylori; His-tag; enzyme kinetics; protein structure; protein stability
Subjects:	NATURAL SCIENCES > Chemistry NATURAL SCIENCES > Biology > Biochemistry and Molecular Biology
Divisions:	Division of Organic Chemistry and Biochemistry Division of Physical Chemistry
Projects:	Project title Project leader Project code Project type Enzimi purinskog reciklirajućeg ciklusa iz Helicobacter pylori i Escherichie coli-PSPE Marija Luić IP-2013-11-7423 HRZZ Alosterički komunikacijski putevi u oligomernim enzimima-ALOKOMP/ALOCOMP Zoran Štefanić; Marko Tomin; Aleksandra Maršavelski; Alessandro Pandini; Bojana Dalbelo-Bašić; Agnieszka Bzowska IP-2019-04-6764 HRZZ
Depositing User:	Ivana Vuglec
Date Deposited:	13 Oct 2025 08:36
URI:	http://fulir.irb.hr/id/eprint/9994
DOI:	10.3390/ijms25147613

Actions (login required)

View Item