Synthesis and Analysis of Biochemical and Pharmaceutical Properties of Polysialylated Recombinant Streptokinase Enzyme

Streptokinase (SK, EC 3.4.99.0) is an enzyme produced by beta-haemolytic streptococcus , and catalyzes the conversion of plasminogen to its active and proteolytic form, plasmin. SK is used in medicine as a fibrinolytic enzyme system for dissolving clots in conditions such as heart attacks, ling artery emboli, vein thrombosis, and occlusions of arteries. Despite the impressive use of this thrombolytic drug, its immunogenicity and short biological half-life are major challenges that limit its efficacy in clinical setting. In this communication, we studied the polysialylation of SK with the aim to improve the pharmacokinetics of this drug in medicine. The skc-2 gene from S. equisimilis ATCC 9542 was codon optimized, and produced as a recombinant enzyme in Escherichia coli W3110. Recombinant SK was covalently conjugated to oxidized polysialic acid (PSA; also referred to as colominic acid; CA) via reductive amination in the presence of NaCNBH ₃ . The native and polysialylated variants were compared in terms of structural properties, enzyme kinetics, stability, immunization and biological half-life. The best molecular weight of PSA, optimum molar ratio, incubation time, and temperature for conjugation reaction of PSA to SK were determined to be 10.0 kDa, 200: 1, 24 h and 25°C, respectively. SDS-PAGE analysis revealed a band at 55.0 kDa for conjugated SK which confirmed the conjugation of PSA to SK. The exact molecular weight of SK-10.0 kDa PSA was determined to be 56.5 kDa by MALDI-TOF spectrometry mass which matches the calculated value by SDS-PAGE. Polysialylation induced a decrease in the far UV CD signal, suggesting an increase in the protein alpha helix content. The intrinsic fluorescence intensity of polysialylated SK increased compared to the native version, meaning that stability of SK was increased by immobilizing on PSA polymer, consistent with the CD results. K _m of polysialylated SK was slightly higher than that of native SK, which showed that the attached PSA molecules to the enzyme did not significantly reduce the substrate specificity. Polysialylated SK elicited nearly 63.0% lower antibody production compared to the native variant. Native and polysialylated SKs exhibited plasma half-life of 0.5 and 2.21 h, respectively, implying that the modified variant has a 4.42-fold longer residence time in body. Briefly, comparative studies with native and PSA-conjugated enzymes show that polysialylation can be useful in enhancing the therapeutic efficacy of SK. It is worth emphasizing that this is the first report describing the use of polysialylation technology to improve the pharmaceutical properties of SK.