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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F25%3A00082379" target="_blank" >RIV/00159816:_____/25:00082379 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14310/25:00142671

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0141813025082121" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0141813025082121</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ijbiomac.2025.147655" target="_blank" >10.1016/j.ijbiomac.2025.147655</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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&apos;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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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&apos;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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/LX22NPO5107" target="_blank" >LX22NPO5107: Národní ústav pro neurologický výzkum</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

International Journal of Biological Macromolecules

ISSN

0141-8130

e-ISSN

1879-0003

Svazek periodika

328

Číslo periodika v rámci svazku

Nov 2025

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

10

Strana od-do

147655

Kód UT WoS článku

001576696500001

EID výsledku v databázi Scopus

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