Concentration‐dependent dimerization of staphylokinase variants with engineered surface charges

Staphylokinase (SAK) is a promising third-generation thrombolytic protein, but its clinical potential is limited by immunogenicity and stability concerns. The conformational and colloidal stabilities of four SAK variants-SAK 42D, SAK STAR, and their non-immunogenic derivatives SAK 42D 3A and SAK STAR 3A-were evaluated using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and aggregation kinetics assays. DSC analyses revealed that thermal denaturation of all variants proceeds via two consecutive irreversible steps, with transition parameters strongly dependent on scan rate and protein concentration. SAK STAR variants exhibited markedly exothermic first transitions and reduced scan rate dependence, suggesting stabilization of intermediate states and suppression of aggregation. In contrast, SAK 42D variants exhibited endothermic or weakly exothermic first transitions and a higher aggregation propensity, correlating with reduced conformational stability and formation of less stable dimers. Colloidal stability tests showed that SAK STAR and SAK STAR 3A remained largely aggregation-resistant, whereas SAK 42D and SAK 42D 3A aggregated rapidly at elevated temperatures (>51°C and >38°C, respectively), following apparent second-order kinetics. DLS confirmed concentration-dependent dimerization in all variants, with SAK 42D 3A displaying pronounced polydispersity and instability. We could rationalize this behavior in the context of engineered surface charges. Our results demonstrate that SAK variant stability is shaped by a complex interplay between primary sequence, dimerization behavior, and aggregation propensity, guiding the design of clinically viable thrombolytic agents and their formulations.