Description

• Title:
  Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori
• Creator:
  Mišković, Marija Zora;
  Wojtyś, Marta;
  Winiewska-Szajewska, Maria;
  Wielgus-Kutrowska, Beata;
  Matković, Marija;
  Domazet Jurašin, Darija;
  Štefanić, Zoran;
  Bzowska, Agnieszka;
  Leščić Ašler, Ivana
• Description:
  first_pagesettingsOrder Article Reprints Open AccessArticle Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori by Marija Zora Mišković 1,†,Marta Wojtyś 2,†,Maria Winiewska-Szajewska 2,3ORCID,Beata Wielgus-Kutrowska 2ORCID,Marija Matković 4,Darija Domazet Jurašin 5ORCID,Zoran Štefanić 5ORCID,Agnieszka Bzowska 2,*ORCID andIvana Leščić Ašler 5,*ORCID 1 Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia 2 Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland 4 Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia 5 Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia * Authors to whom correspondence should be addressed. † These authors contributed equally to this work. Int. J. Mol. Sci. 2024, 25(14), 7613; https://doi.org/10.3390/ijms25147613 Submission received: 30 April 2024 / Revised: 5 July 2024 / Accepted: 8 July 2024 / Published: 11 July 2024 (This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function) Downloadkeyboard_arrow_down Browse Figures Versions Notes 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.
• Object availability:
  free access
• Date:
  2024
• Type:
  Article;
  PeerReviewed
• Language:
  English
• Format:
  application/pdf
• Publisher:
  MDPI
• Relation: http://eprints.ibb.waw.pl/2436 ;http://doi.org/10.3390/ijms25147613 ;doi:10.3390/ijms25147613
• Subject:
  Q Science (General)
• Identifier: http://eprints.ibb.waw.pl/2436/1/ijms-25-07613.pdf ;
  Mišković, Marija Zora and Wojtyś, Marta and Winiewska-Szajewska, Maria and Wielgus-Kutrowska, Beata and Matković, Marija and Domazet Jurašin, Darija and Štefanić, Zoran and Bzowska, Agnieszka and 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). p. 7613. ISSN 1422-0067
• Data provider:
  Repozytorium Instytutu Biochemii i Biofizyki Polskiej Akademii Nauk
• Can I use it?:
  seek permission
• Type:
  other