Volume 22, Issue 2 (2019)                   mjms 2019, 22(2): 77-84 | Back to browse issues page

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Rafipour M, Keramati M, Aslani M, Arashkia A, Roohvand F. Evaluation of the α2-Antipalsmin-Resistance of a Domain Exchanged-Chimeric Streptokinase from Two Streptococci Groups. mjms. 2019; 22 (2) :77-84
URL: http://mjms.modares.ac.ir/article-30-27264-en.html
1- Virology & Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
2- Nano Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
3- Microbiology Department, Pasteur Institute of Iran, Tehran, Iran
4- Virology Department, Pasteur Institute of Iran, Tehran, Iran
5- Virology Department, Pasteur Institute of Iran, Tehran, Iran , rfarzin@pasteur.ac.ir
Abstract:   (5571 Views)
Aims: The chimeric domain-exchanged streptokinase (SKch) between two sk genes from groups G and A streptococci (SK2aG88 and SK2bALAB49, respectively) was constructed to evaluate the role of SK-domains (α, β, γ) in α2-antipalsmin-resistance variations of SK.
Materials and Methods: In this experimental study, PCR-amplified genes of streptococci (skg, ska) were cloned into pET26b vector to produce pET26-SKG88 and pET26-SKALAB49. For domain exchange, the amplicon containing β and g domains of SK2bALAB49 was replaced for that of the SK2aG88 within pET-SK2aG88 (pET26-SKch; α2aG88β2bALABγ2bALAB). All constructs were confirmed by restriction analyses/agarose-gel electrophoresis and DNA sequencing, transformed into E.coli Rosetta, and induced by IPTG for protein expression. Proteins were purified by Ni-NTA chromatography, quantified by Bradford method, and analyzed by SDS-PAGE/Western blotting assays. The α2-antipalsmin-resistance was measured by S2251 colorimetric assay for plasminogen activation.
Findings: SDS-PAGE and western blotting results indicated the expression of proteins with the size of 47kD. At the highest concentration of α2-antiplasmin, SK2aG88 remained 80% active, whereas the SK2bALAB49 and SKch retained 55% of their activity.
Conclusion: SKch shows similar activity reduction, indicating the minor role of the α domain compared to β and g domains for α2-antipalsmin-resistance.
Full-Text [PDF 847 kb]   (1012 Downloads)    
Article Type: Original Research | Subject: Biologic Products
Received: 2018/11/18 | Accepted: 2019/02/12

References
1. Lal V. Fibrinolytic drug therapy in the management of intravascular thrombosis, especially acute myocardial infarction-A review. J Pharmacol Clin Res. 2017;2(4):555593. [Link] [DOI:10.19080/JPCR.2017.02.555593]
2. Mohseni J, Kazemi T, Maleki MH, Beydokhti H. A systematic review on the prevalence of acute myocardial infarction in Iran. Heart Views. 2017;18(4):125-32. [Link] [DOI:10.4103/HEARTVIEWS.HEARTVIEWS_71_17]
3. Klegerman ME. Translational initiatives in thrombolytic therapy. Front Med. 2017;11(1):1-19 [Link] [DOI:10.1007/s11684-017-0497-8]
4. Tourani S, Bashzar S, Nikfar Sh, Ravaghi H, Sadeghi M. Effectiveness of tenecteplase versus streptokinase in treatment of acute myocardial infarction: A meta-analysis. Tehran Univ Med J. 2018;76(6): 380-7. [Persian] [Link]
5. Chapin JC, Hajjar KA. Fibrinolysis and the control of blood coagulation. Blood Rev. 2015;29(1):17-24 [Link] [DOI:10.1016/j.blre.2014.09.003]
6. Adivitiya, Khasa YP. The evolution of recombinant thrombolytics: Current status and future directions. Bioengineered. 2017;8(4): 331-58 [Link] [DOI:10.1080/21655979.2016.1229718]
7. Carpenter SL, Mathew P. Alpha2-antiplasmin and its deficiency: fibrinolysis out of balance. Haemophilia. 2008;14(6):1250-4. [Link] [DOI:10.1111/j.1365-2516.2008.01766.x]
8. Coughlin PB. Antiplasmin: The forgotten serpin?. FEBS J. 2005;272(19):4852-7. [Link] [DOI:10.1111/j.1742-4658.2005.04881.x]
9. Roohvand F. Streptokinase for treatment of thrombotic disorders: The end? Or the end of the beginning? Iran Biomed J. 2018;22(3):140-1. [Link]
10. Rezaei M, Baghbani-Arani F, Arabi Mianroodi R. Point mutation in amino acid 263 of streptokinase gene as well as cloning and expression of the cysteine containing mutated protein. Nova Biologica Reperta. 2016;3(3):249-57. [Persian] [Link] [DOI:10.21859/acadpub.nbr.3.3.258]
11. Keramati M, Arabi Mianroodi R, Memarnejadian A, Mirzaie A, Sazvari S, Mehdi Aslani M, Roohvand F. Towards a superior streptokinase for fibrinolytic therapy of vascular thrombosis. Cardiovasc Hematol Agents Med Chem. 2013; 11(3): 218-29 [Link] [DOI:10.2174/187152571103140120103816]
12. Zhang Y, Liang Z, Glinton K, Ploplis VA, Castellino FJ. Functional differences between Streptococcus pyogenes cluster 1 and cluster 2b streptokinases are determined by their β‐domains. FEBS Lett. 2013;587(9):1304-9. [Link] [DOI:10.1016/j.febslet.2013.02.033]
13. Sazonova IY, McNamee RA, Houng A, King S, Hedstrom L, Reed GL. Reprogrammed streptokinases develop fibrin‐targeting and dissolve blood clots with more potency than tissue plasminogen activator. J Thromb Haemost. 2009;7(8):1321-8 [Link] [DOI:10.1111/j.1538-7836.2009.03491.x]
14. Keramati M, Roohvand F, Eslaminejad Z, Mirzaie A, Nikbin VS, Aslani MM. PCR/RFLP-based allelic variants of streptokinase and their plasminogen activation potencies. J FEMS Microbiol Lett. 2012;335(2):79-85. [Link] [DOI:10.1111/j.1574-6968.2012.02640.x]
15. Kalia A, Bessen DE. Natural selection and evolution of streptococcal virulence genes involved in tissue-specific adaptations. J Bacterial. 2004;186(1): 110-21. [Link] [DOI:10.1128/JB.186.1.110-121.2004]
16. McArthur JD, McKay FC, Ramachandran V, Shyam P, Cork AJ, Sanderson-Smith ML, et al. Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation. FASEB J. 2008;22(9):3146-53. [Link] [DOI:10.1096/fj.08-109348]
17. Cook SM, Skora A, Gillen CM, Walker MJ, McArthur JD. Streptokinase variants from Streptococcus pyogenes isolates display altered plasminogen activation characteristics- implications for pathogenesis. Mol Microbial. 2012;86(5):1052-62. [Link] [DOI:10.1111/mmi.12037]
18. Keramati M, Roohvand F, Aslani MM, Khatami S, Aghasadeghi M, Sadat M, et al. Screening, Cloning and Expression of Active Streptokinase from an Iranian Isolate of S. equisimilis Group C in E. coli. Iran J Basic Med Sci. 2013;16(4): 620-7. [Link]
19. Keramati M, Aslani MM, Khatami S, Roohvand F. 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. 2017;39(6): 889-95. [Link] [DOI:10.1007/s10529-017-2310-9]
20. Sambrook J, Russell DW. The condensed protocols from molecular cloning: a laboratory manual. New York: CSHL Press; 2006. [Link]
21. Arabi R, Roohvand F, Norouzian D, Sardari S, Aghasadeghi MR, Khanahmad H, et al. A comparative study on the activity and antigenicity of truncated and full-length forms of streptokinase. Pol J Microbiol. 2011;60(3):243-51. [Link]
22. Wohl RC, Summaria L, Robbins K. Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 degrees C. J Biol Chem. 1980;255(5):2005-13. [Link]
23. Chaudhary A, Vasudha S, Rajagopal K, Komath SS, Garg N, Yadav M, et al. Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site-directed mutagenesis. Protein Sci. 1999;8(12):2791-2805. [Link] [DOI:10.1110/ps.8.12.2791]
24. Sands D, Whitton CM, Longstaff C. International collaborative study to establish the 3rd international standard for streptokinase. J Thromb Haemost. 2004;2(8):1411-5. [Link] [DOI:10.1111/j.1538-7836.2004.00814.x]
25. Mahboubi A, Sadjady SK, Mirzaei Saleh Abadi M, Azadi S, Solaimanian R. Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay. Iran J Pharm Res. 2012;11(4):1087-93. [Link]
26. Hawkey CM, Stafford JL. A standard clot method for the assay of plasminogen activators, anti-activators, and plasmin. J Clin Pathol. 1964;17(2):175-81. [Link] [DOI:10.1136/jcp.17.2.175]
27. Cederholm‐Williams SA, De Cock F, LIjnen HR, Collen D. Kinetics of the reactions between streptokinase, plasmin and α2‐antiplasmin. Eur J Biochem. 1979;100(1):125-32. [Link] [DOI:10.1111/j.1432-1033.1979.tb02040.x]
28. Mundada LV, Prorok M, DeFord ME, Figuera M, Castellino FJ, Fay WP. Structure-function analysis of the streptokinase amino terminus (residues 1-59). J Biol Chem. 2003;278(27):24421-7. [Link] [DOI:10.1074/jbc.M301825200]
29. Rodríguez P, Fuentes P, Barro M, Alvarez JG, Muñoz E, Collen D, Lijnen HR. Structural domains of streptokinase involved in the interaction with plasminogen. Eur J Biochem. 1995;229(1):83-90. [Link] [DOI:10.1111/j.1432-1033.1995.tb20441.x]
30. Gladysheva IP, Sazonova IY, Chowdhry SA, Liu L, Turner RB, Reed GL. Chimerism reveals a role for the streptokinase β-domain in nonproteolytic active site formation, substrate, and inhibitor interactions. J Biol Chem. 2002;277(30): 26846-51. [Link] [DOI:10.1074/jbc.M202999200]

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