Assessing the impact of His-tags on activity and stability of
staphylokinase variants

J 2025

Assessing the impact of His-tags on activity and stability of staphylokinase variants

STULAJTEROVA, Monika; Lubos AMBRO; Dagmar SEDLAKOVA; Michal NEMERGUT; Pavel KOHOUT et al.

Základní údaje

Originální název

Assessing the impact of His-tags on activity and stability of staphylokinase variants

Autoři

Vydání

International Journal of Biological Macromolecules, Amsterdam, ELSEVIER, 2025, 0141-8130

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Nizozemské království

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 8.500 v roce 2024

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/25:00142689

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

ACUTE ISCHEMIC-STROKE; RECOMBINANT STAPHYLOKINASE; HEXAHISTIDINE-TAG; HISTIDINE TAG; PROTEIN; THROMBOLYTICS; SOLUBILITY; ATTACHMENT

Štítky

14314010, 143180, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 24. 1. 2026 08:00, Mgr. Michaela Hylsová, Ph.D.

Anotace

V originále

Staphylokinase (SAK), a potent plasminogen activator, is a promising thrombolytic agent, but its clinical application is limited by immunogenicity and stability concerns. In addition to intrinsic sequence variants, recombinant protein production often introduces affinity tags, such as the N-terminal polyhistidine (His-tag), whose potential effects on protein's biophysical and functional properties remain poorly understood. Here, we systematically investigated the impact of His-tagging on the stability and activity of four SAK variants: wild-type and non-immunogenic (triple-alanine, 3A) forms of two naturally occurring SAK types, SAK STAR and SAK 42D. Thermal and pH stability were assessed using circular dichroism and tryptophan fluorescence spectroscopy, and plasminogen-activating efficiency was evaluated through chromogenic assays. We found that while the His-tag had little effect on thermal stability and only modestly influenced functional activity, it significantly destabilized SAK under acidic conditions, and altered unfolding transitions, indicating the presence of intermediate conformations. Among the tested proteins, SAK STAR demonstrated the best structural and functional robustness, whereas SAK 42D 3A was the least stable and most prone to aggregation. These results highlight the need to assess the biophysical effect of affinity tags and point SAK STAR as the most suitable candidate for next therapeutic development.

Návaznosti

LX22NPO5107, projekt VaV

Název: Národní ústav pro neurologický výzkum

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Národní ústav pro neurologický výzkum, 5.1 EXCELES

857560, interní kód MU
(Kód CEP: EF17_043/0009632)

Název: CETOCOEN Excellence (Akronym: CETOCOEN Excellence)

Investor: Evropská unie, CETOCOEN Excellence, Spreading excellence and widening participation

Citovat

STULAJTEROVA, Monika; Lubos AMBRO; Dagmar SEDLAKOVA; Michal NEMERGUT; Pavel KOHOUT; Stanislav MAZURENKO; Rastislav VARHAC; Alan STRUNGA; Martin TOUL; Zbyněk PROKOP; Jiří DAMBORSKÝ; Maria TOMKOVA a Erik SEDLÁK. Assessing the impact of His-tags on activity and stability of staphylokinase variants. International Journal of Biological Macromolecules. Amsterdam: ELSEVIER, 2025, roč. 328, November 2025, s. 1-10. ISSN 0141-8130. Dostupné z: https://doi.org/10.1016/j.ijbiomac.2025.147655.

@article{2535247,
   author = {Stulajterova, Monika and Ambro, Lubos and Sedlakova, Dagmar and Nemergut, Michal and Kohout, Pavel and Mazurenko, Stanislav and Varhac, Rastislav and Strunga, Alan and Toul, Martin and Prokop, Zbyněk and Damborský, Jiří and Tomkova, Maria and Sedlák, Erik},
   article_location = {Amsterdam},
   article_number = {November 2025},
   doi = {https://doi.org/10.1016/j.ijbiomac.2025.147655},
   keywords = {ACUTE ISCHEMIC-STROKE; RECOMBINANT STAPHYLOKINASE; HEXAHISTIDINE-TAG; HISTIDINE TAG; PROTEIN; THROMBOLYTICS; SOLUBILITY; ATTACHMENT},
   language = {eng},
   issn = {0141-8130},
   journal = {International Journal of Biological Macromolecules},
   title = {Assessing the impact of His-tags on activity and stability of staphylokinase variants},
   url = {https://www.sciencedirect.com/science/article/pii/S0141813025082121?via%3Dihub},
   volume = {328},
   year = {2025}
}

TY  - JOUR
ID  - 2535247
AU  - Stulajterova, Monika - Ambro, Lubos - Sedlakova, Dagmar - Nemergut, Michal - Kohout, Pavel - Mazurenko, Stanislav - Varhac, Rastislav - Strunga, Alan - Toul, Martin - Prokop, Zbyněk - Damborský, Jiří - Tomkova, Maria - Sedlák, Erik
PY  - 2025
TI  - Assessing the impact of His-tags on activity and stability of staphylokinase variants
JF  - International Journal of Biological Macromolecules
VL  - 328
IS  - November 2025
SP  - 1-10
EP  - 1-10
PB  - ELSEVIER
SN  - 01418130
KW  - ACUTE ISCHEMIC-STROKE
KW  - RECOMBINANT STAPHYLOKINASE
KW  - HEXAHISTIDINE-TAG
KW  - HISTIDINE TAG
KW  - PROTEIN
KW  - THROMBOLYTICS
KW  - SOLUBILITY
KW  - ATTACHMENT
UR  - https://www.sciencedirect.com/science/article/pii/S0141813025082121?via%3Dihub
N2  - Staphylokinase (SAK), a potent plasminogen activator, is a promising thrombolytic agent, but its clinical application is limited by immunogenicity and stability concerns. In addition to intrinsic sequence variants, recombinant protein production often introduces affinity tags, such as the N-terminal polyhistidine (His-tag), whose potential effects on protein's biophysical and functional properties remain poorly understood. Here, we systematically investigated the impact of His-tagging on the stability and activity of four SAK variants: wild-type and non-immunogenic (triple-alanine, 3A) forms of two naturally occurring SAK types, SAK STAR and SAK 42D. Thermal and pH stability were assessed using circular dichroism and tryptophan fluorescence spectroscopy, and plasminogen-activating efficiency was evaluated through chromogenic assays. We found that while the His-tag had little effect on thermal stability and only modestly influenced functional activity, it significantly destabilized SAK under acidic conditions, and altered unfolding transitions, indicating the presence of intermediate conformations. Among the tested proteins, SAK STAR demonstrated the best structural and functional robustness, whereas SAK 42D 3A was the least stable and most prone to aggregation. These results highlight the need to assess the biophysical effect of affinity tags and point SAK STAR as the most suitable candidate for next therapeutic development.
ER  -

STULAJTEROVA, Monika; Lubos AMBRO; Dagmar SEDLAKOVA; Michal NEMERGUT; Pavel KOHOUT; Stanislav MAZURENKO; Rastislav VARHAC; Alan STRUNGA; Martin TOUL; Zbyněk PROKOP; Jiří DAMBORSKÝ; Maria TOMKOVA a Erik SEDLÁK. Assessing the impact of His-tags on activity and stability of staphylokinase variants. \textit{International Journal of Biological Macromolecules}. Amsterdam: ELSEVIER, 2025, roč.~328, November 2025, s.~1-10. ISSN~0141-8130. Dostupné z: https://doi.org/10.1016/j.ijbiomac.2025.147655.

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