Abstract
Objective
To gain insights on the degree of heterogeneity and kinetic differences of streptokinase (SK) from group G (SKG) Streptococci compared with standard SK from group C (SKC) and identification of potentially contributing critical residues (hotspots).
Results
DNA and sequencing analyses confirmed the proper construction of all SK encoding vectors (two SKGs and one standard SKC). SDS-PAGE and western blot analyses confirmed the expression and proper purification of the recombinant SKs from E.coli with the expected size of 47 kDa. Kinetic analyses of two SKGs, compared with SKC, showed higher levels of specific [(×103 IU/mg) of 725 and 715 vs. 536] and fibrin-dependent proteolytic activities [Kcat/KM (min−1/µM) of 37 and 30 vs. 23], accompanied by declined fibrin-independent amidolytic activities [Kcat/KM (min−1/mM) of 109 and 84 vs. 113], respectively. Sequence alignments identified 10 novel residual substitutions scattered in SKα (I33F, R45Q, SKG132, A47D, and G55 N), SKβ (N228 K, F287I), and SKγ domains (L335 V, V396A, T403S) of SKGs, as potential hotspots.
Conclusion
The residue substitutions identified might critically contribute as hot spots to different kinetic parameters of SKGs and might assist in further elucidation of structure/function relations and rational design of SKs with improved (fibrin-dependent) therapeutic properties.
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Acknowledgements
This work was supported by Pasteur Institute of Iran in partial fulfilment of Ph.D thesis of M.K. in Pharmaceutical Biotechnology program.
Supporting information
Supplementary Table 1—Extracted kinetic data from literature for random/directed mutations and deletions studies on SKC compared to SKGs of the present study.
Supplementary Fig. 1—Schematic presentation of HPG activation mechanism by SK in amidolytic (fibrin-independent) and proteolytic (fibrin-dependent) pathways.
Supplementary Fig. 2—Construction of streptokinase expression plasmids.
Supplementary Figs. 3, 4 and 5—Time course of single stage HPG activation, amidolytic and proteolytic activities (by SKC, SKG88 and SKG132), respectively.
Supplementary Fig. 6—Complete amino acid sequence alignment for SKG88, SKG132 and SKC9542.
Supplementary Fig. 7—Construction of the phylogenetic tree, based on 339 bp hyper variable region of SKβ domain (sk-V1; residues 147–218).
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Keramati, M., Aslani, M.M., Khatami, S. et al. Sequence and kinetic analyses of streptokinase from two group G streptococci with high fibrin-dependent plasminogen activities and the identification of novel altered amino acids as potential hot spots. Biotechnol Lett 39, 889–895 (2017). https://doi.org/10.1007/s10529-017-2310-9
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DOI: https://doi.org/10.1007/s10529-017-2310-9