Assessing the Impact of His-tags on Activity and Stability of Staphylokinase Variants
Authors
Stulajterova, M., Ambro, L., Sedlakova, D., Nemergut, M., Kohout, P., Mazurenko, S., Varhac, R., Strunga, A., Toul, M., Prokop, Z., Damborsky, J., Tomkova, M., Sedlak, E.
Source
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 328: 147655 (2025)
Abstract
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, 3 A) 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 3 A 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.