Evaluation of Activity Kinetic Parameters of SK319cys, As a New Cysteine Variant of Streptokinase: A Comparative Study

Author(s): Narges N. Alinodehi, Sanaz Sadeh, Farahnaz K. Nezamiha, Malihe Keramati, Mehdi Hasanzadeh, Reza A. Mianroodi*.

Journal Name: Current Pharmaceutical Biotechnology

Volume 20 , Issue 1 , 2019

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Graphical Abstract:


Background: Despite the extensive use of streptokinase in thrombolytic therapy, its administration may have some shortcomings like allergic reactions and relatively low half life. Specific PEGylation on cysteine at desired sites of streptokinase may alleviate these deficiencies and improve the quality of treatment.

Objective: This study was carried out to create a new cystein variant of streptokinase and compare its activity with formerly mutated SK263cys, SK45cys and intact streptokinase (Ski) to introduce superior candidates for specific PEGylation.

Method: In silico study was carried out to select appropriate amino acid for cysteine substitution and accordingly mutagenesis was carried out by SOEing PCR. The mutated gene was cloned in E. coli, expressed, and purified by affinity chromatography. Activity of the purified proteins was assayed and kinetic parameters of enzymatic reaction were analyzed.

Results: According to in silico data, Arginine319 was selected for substitution with cysteine. SK319cys was achieved with 98% purity after cloning, expression and purification. It was shown that the enzymatic efficiency of SK319Cys and SK263cys was increased 18 and 21%, respectively, when compared to SKi (79.4 and 81.3 vs. 67.1µM-1min-1), while SK45cys showed 7% activity decrease (62.47µM-1min-1) compared to SKi. According to time-based activity assay, SK319Cys and SK263cys exhibited higher activity at lower substrate concentrations (100 and 200 µM), but at higher concentrations of substrate (400 and 800 µM), the proteins showed a very close trend of activity.

Conclusion: SK319cys, as the new cysteine variant of streptokinase, together with SK263cys and SK45cys can be considered as appropriate molecules for specific PEGylation.

Keywords: Streptokinase, mutation, cysteine substitution, cloning, activity assay, expression.

Banerjee, A.; Chisti, C.; Banerjee, U.C. Streptokinase-a clinically useful thrombolytic agent. Biotechnol. Adv., 2004, 22, 287-307.
Collen, D. Coronary thrombolysis: Streptokinase or recombinant tissue-type plasminogen activator? Ann. Intern. Med., 1990, 112(7), 529-538.
Chauhan, S.; Meena, S. Pegylation - a surprising technology. Int. Pharmaceut. Sciencia, 2011, 1(2), 18-24.
Keramati, M.; Arabi Mianroodi, R.; Memarnejadian, A.; Mirzaie, A.; Sazvari, S.; Aslani, M.M.; Roohvand, F. Towards a superior streptokinase for fibrinolytic therapy of vascular thrombosis. Cardiovasc. Hematol. Agents Med. Chem., 2013, 11(3), 218-229.
Cardoso, C.D. Jr., Perobelli, R.F.; Xavier, B.; Maldaner, F.P.; da Silva, F.S.; Dalmora, S.L. Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay. J. Sep. Sci., 2017, 40(2), 407-414.
Huish, S.; Thelwell, C.; Longstaff, C. Activity regulation by fibrinogen and fibrin of streptokinase from Streptococcus pyogenes. PLoS One, 2017, 12(1), e0170936.
Mukhametova, L.I.; Aisina, R.B.; Zakharyan, E.M.; Karakhanov, E.A.; Gershkovich, K.B.; Varfolomeyev, S.D. Thrombolytic and fibrinogenolytic properties of bioconjugate streptokinase-polyamidoamine dendrimers in vitro. Thromb. Res., 2017, 154, 50-52.
Yaghoobi, N.; Faridi, M.R.; Faramarzi, M.A.; Baharifar, H.; Amani, A. Preparation, optimization and activity evaluation of PLGA/streptokinase nanoparticles using electrospray. Adv. Pharm. Bull., 2017, 7(1), 131-139.
Azdaki, N.; Mohammadifard, M.; Ghasemian, M.R.; Saburi, A. Successful intravenous streptokinase therapy in refractory extensive deep vein thrombosis associated with inferior vena cava agenesis: A novel treatment for a rare case. Ann. Card. Anaesth., 2017, 20(2), 268-269.
Baruah, D.B.; Rajendra, N.D.; Chaudhari, M.R.; Kadam, S.S. Plasminogen activators: A comparison. Vascul. Pharmacol., 2006, 44, 1-9.
Cox, G.N.; Smith, D.J.; Carlson, S.J.; Bendele, A.M.; Chlipala, E.A.; Doherty, D.H. Enhanced circulating half-life and hematopoietic properties of a human granulocyte colony-stimulating factor/immunoglobulin fusion protein. Exp. Hematol., 2004, 32(5), 441-449.
Doherty, D.H.; Rosendahl, M.S.; Smith, D.J.; Hughes, J.M.; Chlipala, E.A.; Cox, G.N. Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor. Bioconjug. Chem., 2005, 16(5), 1291-1298.
Kumar, S.; Maheshwari, N.; Shani, G. Mutants of streptokinase and their covalently modified forms. U.S. Patent 8,093,032 B2, 2012.
Parhami-seren, B.; Lynch, M.; Whites, H.D.; Reed, G.L. Mapping the antigenic regions of streptokinase in humans before and after streptokinase therapy. Mol. Immunol., 1995, 32(10), 111-124.
Rajagopalan, S.; Gonias, S.L.; Pizzo, S.V. A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function. J. Clin. Invest., 1985, 75, 413-419.
Torrens, I.; Ojalvo, A.G.; Seralenam, A.; Hayes, O.; Fuente, J. A mutant streptokinase lacking the C-terminal 42 amino acids is less immunogenic. Immunol. Lett., 1999, 70, 213-218.
Brucato, F.H.; Pizzo, S.V. Catabolism of streptokinase and polyethylene glycol-streptokinase: evidence for transport of intact forms through the biliary system in the mouse. Blood, 1990, 76, 73-79.
Lee, J.I.; Eisenberg, S.P.; Rosendahl, M.S.; Chlipala, E.A.; Brown, J.D.; Doherty, D.H.; Cox, G.N. Site-specific PEGylation enhances the pharmacokinetic properties and antitumor activity of interferon beta-1b. J. Interferon Cytokine Res., 2013, 33(12), 769-777.
Sawhney, P.; Katare, K.; Sahni, G. pegylation of truncated streptokinase leads to formulation of a useful drug with ameliorated attributes. PLoS One, 2016, 11(5), e0155831.
Sawhney, P.; Kumar, S.; Maheshwari, N.; Guleria, S.S.; Dhar, N.; Kashyap, R.; Sahni, G. Site-specific thiol-mediated pegylation of streptokinase leads to improved properties with clinical potential. Curr. Pharm. Des., 2016, 22(38), 5868-5878.
Koide, A.; Suzuki, S.; Kobayashi, S. Preparation of polyethylene glycol-modified streptokinase with disappearance of binding ability towards anti-serum and retention of activity. FEBS Lett., 1982, 143(1), 73-76.
Rosendahl, M.S.; Doherty, D.H.; Smith, D.J.; Carlson, S.J.; Chlipala, E.A.; Cox, G.N. A long-acting, highly potent interferon alpha-2 conjugate created using site-specific PEGylation. Bioconjug. Chem., 2005, 16(1), 200-207.
Wang, X.; Lin, X.; Lay, J.A.; Tang, J.; Zhang, X.C. Crystal structure of the catalytic domain of human plasmin complexed with streptokinase. Science, 1998, 281, 1662-1665.
Sambrook, J.; Russell, D. Molecular cloning: a laboratory manual, 3rd ed; Cold Spring Harbor Laboratory Press: New York, 2001.
Arabi, R.; Roohvand, F.; Norouzian, D.; Sardari, S.; Aghasadeghi, M.; Khanahmad, H.; Memarnejadian, A.; Motevalli, F. A comparative study on the activity and antigenicity of truncated and full-length forms of streptokinase. Pol. J. Microbiol., 2011, 60, 243-251.
QIAGEN. The QIAexpressionist. A handbook for high-level expression and purification of 6xHis-tagged proteins, 3rd ed; Hilden: Germany, 2000.
Reed, G.L.; Houng, A.K.; Liu, L.; Parhami-Seren, B.; Matsueda, L.H.; Wang, S.; Hedstrom, L. A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator. Proc. Natl. Acad. Sci., 1999, 96(16), 8879-8883.
Parhami-Seren, B.; Seaveya, M.; Krudysza, J.; Tsantili, P. Structural correlates of a functional streptokinase antigenic epitope: serine 138 is an essential residue for antibody binding. J. Immunol. Methods, 2003, 272, 93-105.
Reed, G.; Kussie, P.; Parhami-seren, B. A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments. J. Immunol., 1993, 150(10), 4407-4415.
Reed, G.L.; Kussie, P.; Parhami-Seren, B. A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments. J. Immunol., 1993, 150(10), 4407-4415.
Coffey, J.A.; Jennings, K.R.; Dalton, H. New antigenic regions of streptokinase are identified by affinity directed mass spectrometry. Eur. J. Biochem., 2001, 268, 5215-5221.

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Year: 2019
Page: [76 - 83]
Pages: 8
DOI: 10.2174/1389201020666190208155808
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