靶向溶栓抗凝双功能融合蛋白的构建表达及在血栓疾病治疗中的应用研究
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摘要
心脑血管血栓性疾病是人类的近年面临的头号杀手。溶栓和抗凝药物组合应用是临床治疗血栓疾病主要给药方案,出血倾向是血栓治疗的主要副作用。为提高葡激酶的选择性、降低葡激酶的临床用药量,同时,降低水蛭素在非血栓部位的浓度或活性从而减小出血的危险,我们利用水蛭素对凝血酶的高亲和性和N—末端延伸引起活性降低的特性,首次设计并构建了通过含有凝血因子FXa识别的六肽连接的葡激酶与水蛭素的融合蛋白,使之具有靶向溶栓抗凝双功能和减小出血倾向等特性。
融合蛋白在毕赤酵母中表达时,葡激酶因第28位天冬酰氨被糖基化而失去活化纤溶酶原活性。Val_(29)突变为Pro的突变体(V29P)基因构建到pPIC9K载体并在甲醇利用型毕赤酵母中得到高效分泌表达,甲醇诱导96h,摇瓶发酵表达量达到1g/L,发酵上清液目的蛋白含量占总蛋白的70%以上,第28位天冬酰氨不发生糖基化;但表达产物不可逆地丧失葡激酶的活化纤溶酶原活性。研究还表明,葡激酶的Asn28、Thr30分别突变为Gln、Ile的突变体均完全丧失活性。计算分析表明,所突变的三个氨基酸使得葡激酶的β折叠结构受到破坏,影响了葡激酶与纤溶酶原的结合,从而使之活性丧失,同时表明SAK的24至30位氨基酸残基形成的β折叠结构对维持其活性和功能是必需的。
将融合蛋白基因克隆到pBV220载体并在大肠杆菌中得到了高效表达。融合蛋白以可溶性蛋白的形式存在与大肠杆菌的细胞质中,采用反复冻融提取、离子交换和凝胶过滤方法纯化了目的蛋白,纯度达96%以上。① 融合蛋白对凝血酶具有一定的靶向性② 完整的融合蛋白具有显著的溶栓功能,丧失抗凝活性,从而可减小出血倾向;但融合蛋白在FXa作用下发生裂解,从而释放出游离水蛭素具有抗凝功能,表明融合蛋白可在血栓部位具有靶向溶栓抗凝双功能。③ 采用卡拉胶诱导的小鼠尾部血栓动物模型实验表明,融合蛋白组小鼠尾部血栓较葡激酶组明显降低,同时尾部血栓形成频率也明显减少,表明融合蛋白具有更好的抗血栓效果。融合蛋白具有良好的应用开发前景。
Despite successful lytic therapy of thromboembolic disorder, reocclusion of the damaged vessels or bleeding complication frequently reduces the therapeutic effect. Making efforts to combine the benefits of thrombolytics and anticoagulants for prevention of vessel reocclusion and to alleviate their side effect of bleeding complication, we designed a targeting bifunctional fusion protein, termed as SFH (Staphylokinase with hirudin linked by FXa recognition peptide).A fusion protein of staphylokinase with hirudin was expressed in methylotrophic yeast Pichia pastoris. The fusion protein yield was up to 1 g/L broth, which expression was induced by fed-batch methanol for 120 hours. Considering the effect of glycosylation of Asn28 of staphylokinase on its activity of activating plasminogen, and to remove the sequence of glycosylation, mutants were constructed and expressed. It was observed that the mutation of amino acid 28-30 dramatically decreased the fibrinolytic of staphylokinase. It was concluded that the fusion protein could be expressed at high level in Pichia pastoris and that the residues of 26-30 amino acid played key role for staphylokinase activating plasminogen.To overcome the dilemma between glycosylation and mutation, the fusion protein without mutation was constructed and expressed in E.coli. The fusion protein retained plasminogen-activating and fibrinolytic activity from the domain of staphylokinase but no anticoagulant activity due to the extension of the N-terminus of hirudin. However, the fusion protein showed effectively anticoagulant activity when fresh thrombus and activated FXa were present due to the local liberation of intact hirudin by FXa. At equimolar concentrations, an enhanced higher anticoagulant activity of the fusion protein than staphylokinase was observed in experiments in a mouse tail thrombosis model induced by kappa-carrageenin. Therefore, the fusion protein SFH is a targeting-released bifunctional molecule being able both to activate fibrinolysis via plasminogen activation and to inhibit clot growth via direct thrombin inhibition at thrombus where the inhibition activity is required. In conclusion, the newly designed chimeric protein has three advantages: targeting to thrombin, fibrinolytic and anticoagulant bifunction, and minimal anticoagulant activity at thrombus-free sites, maximal local concentration and anticoagulant activity in the vicinity of thrombus. It was showed that the fusion protein was a promising drug candidate for targeted therapy of thrombosis.
融合蛋白在毕赤酵母中表达时,葡激酶因第28位天冬酰氨被糖基化而失去活化纤溶酶原活性。Val_(29)突变为Pro的突变体(V29P)基因构建到pPIC9K载体并在甲醇利用型毕赤酵母中得到高效分泌表达,甲醇诱导96h,摇瓶发酵表达量达到1g/L,发酵上清液目的蛋白含量占总蛋白的70%以上,第28位天冬酰氨不发生糖基化;但表达产物不可逆地丧失葡激酶的活化纤溶酶原活性。研究还表明,葡激酶的Asn28、Thr30分别突变为Gln、Ile的突变体均完全丧失活性。计算分析表明,所突变的三个氨基酸使得葡激酶的β折叠结构受到破坏,影响了葡激酶与纤溶酶原的结合,从而使之活性丧失,同时表明SAK的24至30位氨基酸残基形成的β折叠结构对维持其活性和功能是必需的。
将融合蛋白基因克隆到pBV220载体并在大肠杆菌中得到了高效表达。融合蛋白以可溶性蛋白的形式存在与大肠杆菌的细胞质中,采用反复冻融提取、离子交换和凝胶过滤方法纯化了目的蛋白,纯度达96%以上。① 融合蛋白对凝血酶具有一定的靶向性② 完整的融合蛋白具有显著的溶栓功能,丧失抗凝活性,从而可减小出血倾向;但融合蛋白在FXa作用下发生裂解,从而释放出游离水蛭素具有抗凝功能,表明融合蛋白可在血栓部位具有靶向溶栓抗凝双功能。③ 采用卡拉胶诱导的小鼠尾部血栓动物模型实验表明,融合蛋白组小鼠尾部血栓较葡激酶组明显降低,同时尾部血栓形成频率也明显减少,表明融合蛋白具有更好的抗血栓效果。融合蛋白具有良好的应用开发前景。
Despite successful lytic therapy of thromboembolic disorder, reocclusion of the damaged vessels or bleeding complication frequently reduces the therapeutic effect. Making efforts to combine the benefits of thrombolytics and anticoagulants for prevention of vessel reocclusion and to alleviate their side effect of bleeding complication, we designed a targeting bifunctional fusion protein, termed as SFH (Staphylokinase with hirudin linked by FXa recognition peptide).A fusion protein of staphylokinase with hirudin was expressed in methylotrophic yeast Pichia pastoris. The fusion protein yield was up to 1 g/L broth, which expression was induced by fed-batch methanol for 120 hours. Considering the effect of glycosylation of Asn28 of staphylokinase on its activity of activating plasminogen, and to remove the sequence of glycosylation, mutants were constructed and expressed. It was observed that the mutation of amino acid 28-30 dramatically decreased the fibrinolytic of staphylokinase. It was concluded that the fusion protein could be expressed at high level in Pichia pastoris and that the residues of 26-30 amino acid played key role for staphylokinase activating plasminogen.To overcome the dilemma between glycosylation and mutation, the fusion protein without mutation was constructed and expressed in E.coli. The fusion protein retained plasminogen-activating and fibrinolytic activity from the domain of staphylokinase but no anticoagulant activity due to the extension of the N-terminus of hirudin. However, the fusion protein showed effectively anticoagulant activity when fresh thrombus and activated FXa were present due to the local liberation of intact hirudin by FXa. At equimolar concentrations, an enhanced higher anticoagulant activity of the fusion protein than staphylokinase was observed in experiments in a mouse tail thrombosis model induced by kappa-carrageenin. Therefore, the fusion protein SFH is a targeting-released bifunctional molecule being able both to activate fibrinolysis via plasminogen activation and to inhibit clot growth via direct thrombin inhibition at thrombus where the inhibition activity is required. In conclusion, the newly designed chimeric protein has three advantages: targeting to thrombin, fibrinolytic and anticoagulant bifunction, and minimal anticoagulant activity at thrombus-free sites, maximal local concentration and anticoagulant activity in the vicinity of thrombus. It was showed that the fusion protein was a promising drug candidate for targeted therapy of thrombosis.
引文
Arnout J, Simoons M, de Bono D, Rapold HJ, Collen D, Verstraete M. Correlation between level of heparinization and patency of the infarct-related coronary artery after treatment of acute myocardial infarction with alteplase (rt-PA). J Am Coll Cardiol 1992; 20:513-9.
Astrup T, Permin PM. Fibrinolysis in animal organism. Nature 1947; 159:681-2.
Badimon L, Badimon JJ, Lassila R, Heras M, Chesebro JH, Fuster V. Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model: effects of hirudin, heparin, and calcium chelation. Blood 1991; 78:423-34
Bar-Shavit R, Eldor A, Vlodavsky I. Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties. J Clin Invest 1989;84:1096-l 104.
Becker RC, Corrao JM, Ball SP, Gore JM. A comparison of heparin strategies after thrombolytic therapy. Am Heart J 1993;126:750-2.
Bleich SD, Nichols TC, Schumacher RR, Cooke DH, Tate DA, Teichman SL. Effect of heparin on coronary arterial patency after thrombolysis with tissue plasminogen activator in acute myocardial infarction. Am J Cardiol 1990;66:1412-7.
Brugemann J. Thrombolysis in acute myocardial infarction factors determining its efficacy. PhD Thesis. 1994.
Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. PNAS. 100(9): 5022-5027, 2003.
Collen D. Identification and some properties of a new fast reacting plasmin inhibitor in human plasma. Eur J Biochem 1976;69:209-16.
Collen D. On the regulation and control of fibrinolysis. Edward Kowalski Memorial Lecture. Thromb Haemost. 1980; 43:77-89.
Collen D. Staphylokinase: a potent, uniquely fibrin-selective thrombolytic agent. Mature Medicine 1998, 4(3): 279-284.
Coller BS, Scudder LE. Inhibition of dog platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antibody. Blood 1986; 66:1456-9.
Coller BS. Platelets and thrombolytic therapy. N Engl J Med 1990;322:33-42.
de Bono DP, et al. for the European Cooperative Study Group. Effect of early intravenous heparin on coronary patency, infarct size, and bleeding complications after alteplase thrombolysis: a result of a randomized double blind European Cooperative Study Group trial. Br Heart J 1992;67:122-8
Deutsch E, Rao AK, Colman RW. Selective thrombin inhibitors: The next generation of anticoagulants. J Am Coll Cardiol 1993;22:1089-92.
European Working Party. Streptokinase in acute myocardial infarction. Br Med J 1971;3:325-31.
Fletcher AP, Alkjaersig N, Sherry S. The maintenance of a sustained thrombolytic state in man. I. Induction a'n d effects. J Clin Invest 1959;38:1096-1110.
Fletcher AP, Alkjaersig N, Smyrniotis FE, Sherry S. Treatment of patients suffering from early, myocardial infarction with massive and prolonged streptokinase therapy. Trans Assoc Am Physicians 1958;71:287-96.
Fletcher AP, Sherry S, Alkjaersig N. The maintenance of a sustained thrombolytic state in man. II.
Clinical observations on patients with myocardial infarction and other thrombo-embolic disorders. J Clin Invest. 1959;38:1111-9.
Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential. Eur J Biochem. 1995; 234: 350-357.
Gallino A, Haeberli A, Hess T, Mombelli G. Fibrin formation and platelet aggregation in patients with acute myocardial infarction: effects of intravenous and subcutaneous low-dose heparin. Am Heart J 1986; 12:285-90.
Gash A, Spann JF, Sherry S, et al. Factors influencing reocclusion after coronary thrombolysis for acute myocardial infarction. Am J Cardiol 1986;57:175-7.
Gold HK, Torres FW, Garabedian HD, et al. Evidence for a rebound coagulation phenomenon after cessation of a 4-hour infusion of a specific thrombin inhibitor in patients with unstable angina pectoris. J Am Coll Cardiol 1993; 21:1039-47.
Goldhaber SZ. Conjunctive heparin therapy. Circulation 1992;86:1639-41.
Grill HP, Spero JE, Granato JE. Comparison of activated partial thromboplastin time to activated clotting time for adequacy of heparin anticoagulation just before percutaneous transluminal coronary angioplasty. Am J Cardiol 1993;71:1219-20.
Grutter MG, Priestle JP, Rahuel J, et al (1990). Crystal structure of the thrombin-hirudin complex: novel model of serine protease inhibition. EMBO J. 9: 2361-2365. Ridker PM, Hebert PR, Fuster V, Hennekens CH. Are both aspirin and heparin justified as adjuncts to thrombolytic therapy for myocardial infarction. Lancet 1993;341:1574-7.
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Badimon L, Badimon JJ, Lassila R, Heras M, Chesebro JH, Fuster V. Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model: effects of hirudin, heparin, and calcium chelation. Blood 1991; 78:423-34
Bar-Shavit R, Eldor A, Vlodavsky I. Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties. J Clin Invest 1989;84:1096-l 104.
Becker RC, Corrao JM, Ball SP, Gore JM. A comparison of heparin strategies after thrombolytic therapy. Am Heart J 1993;126:750-2.
Bleich SD, Nichols TC, Schumacher RR, Cooke DH, Tate DA, Teichman SL. Effect of heparin on coronary arterial patency after thrombolysis with tissue plasminogen activator in acute myocardial infarction. Am J Cardiol 1990;66:1412-7.
Brugemann J. Thrombolysis in acute myocardial infarction factors determining its efficacy. PhD Thesis. 1994.
Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. PNAS. 100(9): 5022-5027, 2003.
Collen D. Identification and some properties of a new fast reacting plasmin inhibitor in human plasma. Eur J Biochem 1976;69:209-16.
Collen D. On the regulation and control of fibrinolysis. Edward Kowalski Memorial Lecture. Thromb Haemost. 1980; 43:77-89.
Collen D. Staphylokinase: a potent, uniquely fibrin-selective thrombolytic agent. Mature Medicine 1998, 4(3): 279-284.
Coller BS, Scudder LE. Inhibition of dog platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antibody. Blood 1986; 66:1456-9.
Coller BS. Platelets and thrombolytic therapy. N Engl J Med 1990;322:33-42.
de Bono DP, et al. for the European Cooperative Study Group. Effect of early intravenous heparin on coronary patency, infarct size, and bleeding complications after alteplase thrombolysis: a result of a randomized double blind European Cooperative Study Group trial. Br Heart J 1992;67:122-8
Deutsch E, Rao AK, Colman RW. Selective thrombin inhibitors: The next generation of anticoagulants. J Am Coll Cardiol 1993;22:1089-92.
European Working Party. Streptokinase in acute myocardial infarction. Br Med J 1971;3:325-31.
Fletcher AP, Alkjaersig N, Sherry S. The maintenance of a sustained thrombolytic state in man. I. Induction a'n d effects. J Clin Invest 1959;38:1096-1110.
Fletcher AP, Alkjaersig N, Smyrniotis FE, Sherry S. Treatment of patients suffering from early, myocardial infarction with massive and prolonged streptokinase therapy. Trans Assoc Am Physicians 1958;71:287-96.
Fletcher AP, Sherry S, Alkjaersig N. The maintenance of a sustained thrombolytic state in man. II.
Clinical observations on patients with myocardial infarction and other thrombo-embolic disorders. J Clin Invest. 1959;38:1111-9.
Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential. Eur J Biochem. 1995; 234: 350-357.
Gallino A, Haeberli A, Hess T, Mombelli G. Fibrin formation and platelet aggregation in patients with acute myocardial infarction: effects of intravenous and subcutaneous low-dose heparin. Am Heart J 1986; 12:285-90.
Gash A, Spann JF, Sherry S, et al. Factors influencing reocclusion after coronary thrombolysis for acute myocardial infarction. Am J Cardiol 1986;57:175-7.
Gold HK, Torres FW, Garabedian HD, et al. Evidence for a rebound coagulation phenomenon after cessation of a 4-hour infusion of a specific thrombin inhibitor in patients with unstable angina pectoris. J Am Coll Cardiol 1993; 21:1039-47.
Goldhaber SZ. Conjunctive heparin therapy. Circulation 1992;86:1639-41.
Grill HP, Spero JE, Granato JE. Comparison of activated partial thromboplastin time to activated clotting time for adequacy of heparin anticoagulation just before percutaneous transluminal coronary angioplasty. Am J Cardiol 1993;71:1219-20.
Grutter MG, Priestle JP, Rahuel J, et al (1990). Crystal structure of the thrombin-hirudin complex: novel model of serine protease inhibition. EMBO J. 9: 2361-2365. Ridker PM, Hebert PR, Fuster V, Hennekens CH. Are both aspirin and heparin justified as adjuncts to thrombolytic therapy for myocardial infarction. Lancet 1993;341:1574-7.
Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, et al. Production of Complex Human Glycoproteins in Yeast. Science 301 (8): 1244-1246, 2003.
Harker LA, Mann KG. Thrombosis and fibrinolysis. In: Fuster V, Verstraete M, ed. Thrombosis in cardiovascular disorders. Philadelphia: Saunders, 1992:1-16.
Hasslacher M, Schall M, Hayn M, et al (1997). High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevvea brasiliensis. Protein Expression and Purification. 11:61-71.
Hirsh J. Heparin. N Engl J Med 1991;324:1565-74.
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