Molecular cloning of Reteplase and its expression in E. coli using tac promoter

Authors
Safieh Aghaabdollahian 1
Mohammad Rabbani 1
Kamran Ghaedi 2
Hamid Mir Mohammad Sadeghi 1
1 Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran
Abstract
Background and Aims: This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression.
Materials and Methods: Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE.
Results: Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG.
Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli.

Keywords

1. Mattes R. The production of improved tissue-type plasminogen activator in Escherichia coli. Semin Thromb Hemost 2001;27:325-36.  Back to cited text no. 1
    
2. Ellis K, Brener S. New fibrinolytic agents for MI: As effective as current agents, but easier to administer. Cleve Clin J Med 2004;71:20,3-5,9-30.  Back to cited text no. 2
    
3. Davami F, Sardari S, Majidzadeh AK, Hemayatkar M, Barkhordari F, Enayati S, et al. A novel variant of t-PA resistant to plasminogen activator inhibitor-1; Expression in CHO cells based on in silico experiments. BMB Rep 2011;44:34-9.  Back to cited text no. 3
    
4. Mandi N, Sundaram KR, Tandra SK, Bandyopadhyay S, Padmanabhan S. Asn and Asn: Critical residues for in vitro biological activity of reteplase. Adv Hematol 2010;2010:17284.  Back to cited text no. 4
    
5. Simpson D, Siddiqui Ma, Scott Lj, Hilleman DE. Spotlight on reteplase in thrombotic occlusive disorders. Bio Drugs 2007;21:65-86.  Back to cited text no. 5
    
6. Simpson D, Siddiqui MA, Scott LJ, Hilleman DE. Reteplase: A review of its use in the management of thrombotic occlusive disorders. Am J Cardiovasc Drugs 2006;6:265-85.  Back to cited text no. 6
    
7. Manosroi J, Tayapiwatana C, Gotz F, Werner RG, Manosroi A. Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli. Appl Environ Microbiol 2001;67:2657-64.  Back to cited text no. 7
    
8. Bass Sh, Yansura DG. Application of the E. coli trp promoter. Mol Biotechnol. 2000;16:253-60.  Back to cited text no. 8
    
9. Khan SR, Gaines J, Roop RM, Farrand SK. Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ Microbiol 2008;74:5053-62.  Back to cited text no. 9
    
10. Boer H, Comstock LJ, Vasser M. The tac promoter: A functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A 1983;80:21-5.  Back to cited text no. 10
    
11. Mulligan M, Brosius J, McClure WR. Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter. J Biol Chem 1985;260:3529-38.  Back to cited text no. 11
    
12. Bagdasarian MM, Amann E, Lurz R, Ruckert B, Bagdasarian M. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene 1983;26:273-82.  Back to cited text no. 12
    
13. Sadeghi H, Rabbani M, Rismani E, Moazen F, Khodabakhsh F, Dormiani K, et al. Optimization of the expression of reteplase in Escherichia coli. Res Pharm Sci 2011;6:87-92.  Back to cited text no. 13
    
14. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory; 1989.  Back to cited text no. 14
    
15. Gao L, Zhang C, Li L, Liang L, Deng X, Wu W, et al. Construction, expression and refolding of a bifunctional fusion protein consisting of C-terminal 12-residue of hirudin-PA and reteplase. Protein J 2012;31:328-36.  Back to cited text no. 15
    
16. Baruah DB, Dash RN, Chaudhari MR, Kadam SS. Plasminogen activators: A comparison. Vascu Pharmacol 2006;44:1-9.  Back to cited text no. 16
    
17. Rouf SA, Moo-Young M, Chisti Y. Tissue-type plasminogen activator: Characteristics, applications and production technology. Biotechnol Adv 1996;14:239-66.  Back to cited text no. 17
    
18. Ogrydziak DM. Yeast extracellular proteases. Crit Rev Biotechnol 1993;13:1-55.  Back to cited text no. 18
    
19. Furlong Am, Thomsen DR, Marotti KR, Post LE, Sharma SK. Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector. Biotechnol Appl Biochem 1988;10:454-64.  Back to cited text no. 19
    
20. Griffiths Jb, Electricwala A. Production of tissue plasminogen activators from animal cells. Adv Biochem Eng Biotechnol 1987;34:147-66.  Back to cited text no. 20
    
21. Liao JZ, J. Shen Zl. Cloning and Expression of Tissue-Type Plasminogen Activator Mutant Reteplase(r-PA) in E. coli. Pharm Biotech 2002;9:94-8.  Back to cited text no. 21
    
22. Holton TA, Graham MW. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res 1991;19:1156.  Back to cited text no. 22
    
23. Sadeghi HM, Ahmadi R, Aghaabdollahian S, Mofid MR, Ghaemi Y, Abedi D. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene. Res Pharm Sci 2011;6:51-5.  Back to cited text no. 23
    
24. Amann E, Brosius J, Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 1983;25:167-78.  Back to cited text no. 24
    
25. Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem 1991;192:262-7.  Back to cited text no. 25
    
26. Frangioni JV, Neel BG. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 1993;210:179-87.  Back to cited text no. 26
    
27. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 1988;67:31-40.   Back to cited text no. 27
    
28. Khodabakhsh F, Moazen F, Mir Mohammad Sadeghi H, Rabbani M. Expression of reteplase in Escherichia coli top10 using arabinose Promoter. Res Pharm Sci 2012;7:S448.  Back to cited text no. 28
    
29. Aflakiyan S, Sadeghi HM, Shokrgozar M, Rabbani M, Bouzari S, Jahanian-Najafabadi A. Expression of the recombinant plasminogen activator (reteplase) by a non-lytic insect cell expression system. Res Pharm Sci 2013;8:9-15.  Back to cited text no. 29
Advanced Biomedical Research
Volume 2014, september
September 2014
Pages 1-5