Concentration-dependent dimerization of staphylokinase variants with engineered surface charges

Area of Science:

• Biochemistry
• Protein Engineering
• Pharmacology

Background:

• Staphylokinase (SAK) is a third-generation thrombolytic protein with clinical promise.
• Clinical application of SAK is hindered by immunogenicity and stability issues.

Purpose of the Study:

• To evaluate the conformational and colloidal stabilities of four SAK variants.
• To compare the stability of SAK 42D and SAK STAR variants with their non-immunogenic derivatives (SAK 42D 3A and SAK STAR 3A).

Main Methods:

• Differential Scanning Calorimetry (DSC) for thermal denaturation analysis.
• Dynamic Light Scattering (DLS) for assessing dimerization and polydispersity.
• Aggregation kinetics assays to quantify aggregation rates.

Main Results:

• SAK STAR variants demonstrated enhanced thermal stability with exothermic transitions, suggesting stabilized intermediates and suppressed aggregation.
• SAK 42D variants exhibited lower conformational stability, increased aggregation propensity, and formed less stable dimers.
• SAK STAR and SAK STAR 3A showed significant aggregation resistance, while SAK 42D and SAK 42D 3A aggregated rapidly at elevated temperatures.

Conclusions:

• SAK variant stability is influenced by primary sequence, dimerization, and aggregation.
• Engineered surface charges can rationalize observed stability differences.
• Findings guide the development of more stable and clinically viable SAK-based thrombolytic agents.