Point mutation in amino acid 263 of streptokinase gene as well as cloning and expression of the cysteine containing mutated protein

Rezaee, Mahsa; Baghbani Arani, Fahimeh; Arabi Mianroodi, Reza

doi:10.21859/acadpub.nbr.3.3.258

Point mutation in amino acid 263 of streptokinase gene as well as cloning and expression of the cysteine containing mutated protein

Authors

Mahsa Rezaee

Fahimeh Baghbani Arani

Reza Arabi Mianroodi

https://doi.org/10.21859/acadpub.nbr.3.3.258

Abstract

Streptokinase is one of the best known thrombolytic agents with widespread clinical use. However, its use is not risk-free due to its immunogenicity, hemorrhagic complications and relatively short half-life in circulation. Specific PEGylation of cysteine residue is a useful technique for reducing most of these complications. The aim of this study was designing and producing a cysteine containing mutant of streptokinase, to be used for specific PEGylation. Glut-amic acid 263, which is a surface amino acid in the structure of streptokinase protein, was selected for replacement with cysteine amino acid by site directed mutagenesis. The Glu263 codon was changed to cysteine codon by SOEing PCR technique. Then, the intact and mutated streptokinase genes were inserted into expression vector pET-26b (+). The co-nstructs were transformed to Escherichia.coli Rosetta (DE3) strain and the proteins were expressed by IPTG induction. The proteins were confirmed by SDS-PAGE and western blot analysis, purified by Ni-NTA agarose affinity chroma-tography under denaturing condition with urea and Sephadex G-25 column was applied to remove urea to refold the pr-oteins. This study indicated that by using aforesaid vector and host, cysteine containing mutant gene is expressed well and it will be appropriate for specific PEGylation.

Keywords

thrombolytic

PEGylation

glutamic acid

purification

20.1001.1.24236330.1395.3.3.8.6

Arabi, R., Roohvand, F., Noruzian, D., Aghasadeghi, MR., Memarnejadian, A., Khanahmad, H., Sadat, M. and Mo-tevali, F. 2011. Cloning and expression of truncated and intact streptokinase molecules in E.coli and evaluation of their biological activities. – J. Agricul. Biotech. 2: 55-67.
Banerjee, A., Chisti, Y. and Banerjee, U.C. 2004. Strept-okinase—a clinically useful thrombolytic agent. – Biot-ec. Adv. 22: 287-307.
Baruah, D.B., Dash, R.N., Chaudhari, M.R. and Kadam, S.S. 2005. Plasminogen activators: A comparison. – Va-scul. Pharmacol. 44: 1-9.
Bornhorst, J. and Falke, J.J. 2000. Purification of proteins using polyhistidine affinity tags. – Methods Enzymol. 326: 245-254.
Chauhan, S. and Meena, S. 2011. Pegylation - A Sunrising Technology. – IPS 1: 18-24.

Damodaran, V.B. and Fee, C.J. 2010. Protein PEGylation: An overview of chemistry and process considerations. – Eur. Pharmace. Rev. 15: 18-26.
Del Zoppo, G.J. 2010. Plasminogen activators in ischemic stroke: introduction. – Stroke 41: 39-41.
Fu, W., Lin, J. and Cen, P. 2007. 5-Aminolevulinate pr-od-uction with recombinant Escherichia coli using a rare co-don optimizer host strain. – Appl. Microbiol. Biotechnol. 75: 777-782.
Ghosh, M., Pulicherla, K.K., Rekha, V.P.B., Venkat Rao, G. and Rao, K. 2012. A review on successive generati-ons of streptokinase based thrombolytic agents. – Int. J. Pharm. Sci. 4: 38-42.
Kumar, S., Maheshwari, N. and Sahni, G. 2012. Mutants of streptokinase and their covalently modified forms. – US Patent Number: 8093032 B2.
Monzavi, N., Aghasadeghi, M.R., Arabi, R., Memarnej-adian, A., Sadat, S.M., Khanahmad, H., Ebrahimi, M. and Roohhvand, F. 2010. Design, cloning, expression and evaluation of cysteine-substitutes of intact and trun-cated molecules of streptokinase. – Iran JPP 14: 56-65.
Pimienta, E., Ayala, JC., Rodríguez, C., Ramos, A., Mell-aert, L.V., Vallín, C. and Anné, J. 2007. Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans. – Microb. Cell Fact 6: 1-8.
Pizzo, S.V. 1991. Preparation, in vivo properties and prop-osed clinical use of polyoxyethylene-modified tissue pl-asminogen activator and streptokinase. – ‎Adv. Drug Deliv. Rev. 6: 153-166.
Pratap, J., Rajamohan, G. and Dikshit, K.L. 2000. Char-acteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation. – Appl. Microbiol. Biotechnol. 53: 469-475.
Rachmawati, H.D.F., Darfiansyah, I.A. and Retnonin-grum, D.S. 2014. PEGylation of recombinant mutein st-reptokinase from overproduction in Escherichia coli Bl-21 and study on the fibrinolityc activity in vitro. – Int. J. Pharm. Pharm. Sci. 6: 137-141.
Rajagopalan, S., Pizzo, S.V. and Gonias, L.S. 1985. A no-nantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function. – J. Clin. Invest. 75: 413-419.
Reed, G.L., Houng, A.K., Liu, L., Parhami-Seren, B., Ma-tsueda, L.H., Wang, S. and Hedstrom, L. 1999. A ca-ta-lytic switch and the conversion of streptokinase to a fi-brin-targeted plasminogen activator. – Proc. Natl. Acad. Sci. USA 96: 8879-8883.
Roberts, M.J., Bentley, M.D. and Harris, J.M. 2002. Ch-emistry for peptide and protein PEGylation. – Adv. Dr-ug. Deliv. Rev. 54: 459-476.
Rother, J., Ford, G.A. and Thijs, N.S. 2013. Thrombolytics in acute ischaemic stroke: historical perspective and fu-ture opportunities. – Cerebrovasc. Dis. 35: 313-319.
Sambrook, J. and Russell, D.W. 2001. Molecular Cloning, a laboratory manual. – CSHL Press.
Sikri, N. and Bardia, A. 2007. History of streptokinase use in acute myocardial infarction. – Tex. Heart. Inst. J. 34: 318-327.
Scott, M. 1995. Cysteine-pegylated proteins. – USA Patent Number: 5766897.
Shani, G., kumar, R., Roy, C., Rajagopal, K., Nihalani, D., Sundram, V. and Yadav, M. 2007. Nucleic acid mole-cules encoding clot specific streptokinase fusion proteins proccessing altered plasminogen activation characte-ristics. – USA Patent Number: 7250503 B2.
Vellanki, R.N., Potumarthi, R., Doddapaneni, K.K., An-ubrolu, N. and Mangamoori, L.N. 2013. Constitutive op-timized production of streptokinase in Saccharomyces cerevisiae utilizing glyceraldehyde 3-phosphate dehy-drogenase promoter of Pichia pastoris. – BioMed. Res. Int. 2013: 249-268.
Veronese, F.M. 2001. Peptide and protein PEGylation: a review of problems and solutions. – Biomat. 22: 405-417.
Werner, M.H., Clore, G.M., Gronenborn, A.M., Kondoh, A. and Fisher, R.J. 1994. Refolding proteins by gel filt-ration chromatography. – FEBS Lett. 345: 125-130.
Wu, X.C., Ye, R., Duan, Y. and Wong, S.L. 1998. Engi-neering of plasmin-resistant forms of streptokinase and their production in Bacillus subtilis: streptokinase with longer functional half-life. – Appl. Microbiol. Biotechnol. 64: 824-829.