Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Cloning, Expression, and Renaturation Studies of Reteplase

  • Zhao, Youchun (State Key Laboratory of Microbial Technology, Shandong University) ;
  • Ge, Wang (State Key Laboratory of Microbial Technology, Shandong University) ;
  • Kong, Young (State Key Laboratory of Microbial Technology, Shandong University) ;
  • Zhang, Changkai (State Key Laboratory of Microbial Technology, Shandong University)
  • Published : 2003.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

Recombinant human tissue plasminogen activator deletion mutein (Reteplase) is a clinically promising thrombolytic drug. Reteplase cDNA was subcloned into a bacteria expression system, and the resultant recombinant was biologically characterized. The Reteplase was expressed in Escherichia coli as an inclusion body, and the downstream processes of the Reteplase inclusion body included denaturation, renaturation, and purification. A protein disulfide isomerase (PDI) was used to assist the refolding of Reteplase, and it was found to increase the refolding rate from less than 2％ to more than 20％. The refolded Reteplase was purified through two chromatography steps, including lysine-coupled agarose affinity chromatography and then CM-sepharose cation-exchange chomatography. The purity of r-PA was analyzed by Western bolt analysis, and N-terminal amino acid and amino acid composition analyses confirmed the end-product. Reteplase showed higher thrombolytic potency in an animal thrombus model.

Keywords

References

  1. Blood Coagul. Fibrinolysis v.3 An ELISA for tissue factor using monoclonal antibodies Albrecht,S.;T.Luther;H.Grossman;C.Flossel;M.Kotzsch;M.Muller https://doi.org/10.1097/00001721-199206000-00005
  2. Circulation v.94 Randomized comparison of coronary thrombolysis achieved with double-bolus reteolase (recombinant plasminogen activator) and front-loaded, accelerated alteplase (recombinant tissue plasminogen activator) in patients with acute myocardial infarction. The RAPID Ⅱ Investigation Bode,c.;R.W.Smalling;G.Berg;C.Burnett;G.Lorch;J.M.Kalbfleisch;R.Chernoff;L.G.Christie;R.L.Feldman;A.A.Seals;W.D.Weaver https://doi.org/10.1161/01.CIR.94.5.891
  3. Druge v.38 Tissue-type plasminogen activator: A review of its pharmacology and therapeutic use as a thrombolytic agent Collen,D.;H.R.Lijnen;P.A.Todd;K.L.Goa https://doi.org/10.2165/00003495-198938030-00003
  4. J. Mol. Biol. v.142 Catalysis by protein-disulphide isomerase of the unfolding and refolding of proteins with disu;phide bonds Creighton,T.E.;D.A.Hillson;R.B.Freedman https://doi.org/10.1016/0022-2836(80)90205-3
  5. Symposium on Frontiers of Biotechnology and Pharmaceuticals A novel engineering clot buster-Reteplase papers Wang,G.;Y.Zhao;Y.Kong;W.Wang;B.Li;F.Xue;C.Zhang
  6. Protein Express. Purif. v.2 Catalysis of protein folding by agarose-immobilized protein disufide isomerase Gilbert,H.F.
  7. Biochem. J. v.275 The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulfide-isomerase Hawkins,H.C.;R.B.Freedman https://doi.org/10.1042/bj2750335
  8. Protein Eng. v.5 Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022 Kohnert,U.;R.Rudolph;J.H.Verheijen;E. J. Weening Verhoeff;A.Stern;U.Opiz;U.Martin;H.Lill;H.Prinz;M.Lechner https://doi.org/10.1093/protein/5.1.93
  9. J. Am. Coll. Cardiol. v.19 Evaluation of thrombolytic and systemic effects of the novel recombinatnt plasminogen activator BM06.022 compared with alteplase, anistreplase, streptokinase in a canine model of coronary artery thrombosis Martin,U.;G.Sponer;K.Strein https://doi.org/10.1016/0735-1097(92)90501-D
  10. Cardiovasc. Drug Rev. v.1 BM06.022: A novel recombinant plasminongen activator Martin,U.;R.Bader;Y.Bohm;Y.Kohner;V.Mollendorf;S.Fisher;G.Sponer
  11. J. Am. Coll. Cardiol. v.24 Dose finding with a novel recombinant plasminogen activator (BM06.022) in patients with acute myocardial infarction: Results of the German Recombinant Plasminogen Activator Study Neuhaus,K.L.;R. von Essen;A.Vogt;V.Atebbe;J.Rustige;H.J.Wagner;K.F.Appel;U.Stienen;R.Konig;W.Meger-Sabelek https://doi.org/10.1016/0735-1097(94)90541-X
  12. Drugs v.52 A review of its pharmacological properties and clinical efficacy in the management of acute myocardial infarction Noble,S.;D.McTavish
  13. J. Biol. Chem. v.267 Protein disulfide isomerase. A multifunctional protein resident in the lumen of the endoplasmic reticulum Novia,R.;W.J.Lennarz
  14. Thromb. Haemost. v.72 In vitro stability of a tissue-type plasminogen activator mutant, BM06.022 Rijken,D.C.;E.Groeneveld;M.M.Barrett-Bergshoeff
  15. Ann. N.Y. Acad. Sci. v.4 Bolus application of a novel recombinant plasminogen activator in acute myocardial infarction patients: Pharmacokinetics and effects on the hemostatic system Seifried,E.;M.M.Muller;U.Martin;R.Konig;V.Hombach
  16. AM. J. Health Syst. Pharm. v.54 A fresh look at the molecular pharmacology of plasminogen activators: From theory to test tube to clinical outcomes Smalling,R.W.
  17. Pharmacotheraph v.16 Tutorial biochemical and pharmacologic comparison of thrombolytic agents Stringer,K.A.
  18. Biochem. J. v.301 Assisted refolding of recombinant prochymosin with the aid of protein disulphide isomerase Tang,B.;S.Zhang;K.Yang
  19. Thromb. Haemost. v.48 A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma Verheijen,J.H.;E.Mullart;G.T.Chang;C.Klu;G.Wijngaards
  20. Drug Evaluation v.50 A reappraisal of its pharmacology and therapeutic use in vascular disorders other acute myocardial infarction Wagstaff,A.J.;J.C.Gillis;K.L.A.Goa
  21. Sci. Technol. Chem. Ind. v.11 Confirm of biochemical properties of reteplase Zhao,Y.;G.Wang;C.Zhang