单修饰重组水蛭素的设计, 制备及体外生物活性测定

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单修饰重组水蛭素的设计, 制备及体外生物活性测定

侯蓓蓓; 李世荣; 李晓晖; 修志龙

Department of Bioscience & Biotechnology, Dalian University of Technology, Dalian 116024, China

收稿日期:2006-12-26 修回日期:1900-01-01 出版日期:2007-12-28 发布日期:2007-12-28
通讯作者: 侯蓓蓓

Design, preparation and in vitro bioactivity of mono-PEGylated recombinant hirudin

HOU Beibei; LI Shirong; LI Xiaohui; XIU Zhilong

Department of Bioscience & Biotechnology, Dalian University of Technology, Dalian 116024, China

Received:2006-12-26 Revised:1900-01-01 Online:2007-12-28 Published:2007-12-28
Contact: HOU Beibei

摘要/Abstract

摘要： Hirudin, the most potent inhibitor of thrombin found in nature, has a short half-life in serum, which significantly limits its clinical application as an anticoagulant. Recently, PEGylation has been commonly used as an effective method to prolong its half-life in serum. In contrast to the nonspecific PEGylation under basic conditions that targets lysine residues randomly, PEGylation sites under mildly acidic conditions preferably targets histidine residues, and there is only one histidine residue at 51 in r-hirudin; therefore, succinimidyl carbonyl methoxy poly-ethylene glycol (SC-mPEG, 20000) was attached to r-hirudin at mildly acidic pH to favor the formation of mono- PEGylated r-hirudin. The reaction mixture with high mono-PEGylated ratio was easily separated by a one-step ion-exchange chromatographic (IEC) procedure. Approximately 79.71% of the mono-PEGylated r-hirudin was PEGylated at His51, which showed that the acidic PEGylation operation prevented the PEGylation of active center (Lys47) of r-hirudin in principle. Mono-PEGylated product with purity higher than 95% was obtained as the pre-dominant product, and 34% of the anticoagulant activity was retained in vitro. The staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was improved to obtain perfect electrophoretic pattern in less than 5min. More accurate molecular weight was deduced due to the use of PEGs as molecular weight standards.

关键词: r-hirudin;histidine residues;SC-mPEG;mono-PEGylation;pH;anticoagulant activity

Abstract: Hirudin, the most potent inhibitor of thrombin found in nature, has a short half-life in serum, which significantly limits its clinical application as an anticoagulant. Recently, PEGylation has been commonly used as an effective method to prolong its half-life in serum. In contrast to the nonspecific PEGylation under basic conditions that targets lysine residues randomly, PEGylation sites under mildly acidic conditions preferably targets histidine residues, and there is only one histidine residue at 51 in r-hirudin; therefore, succinimidyl carbonyl methoxy poly-ethylene glycol (SC-mPEG, 20000) was attached to r-hirudin at mildly acidic pH to favor the formation of mono- PEGylated r-hirudin. The reaction mixture with high mono-PEGylated ratio was easily separated by a one-step ion-exchange chromatographic (IEC) procedure. Approximately 79.71% of the mono-PEGylated r-hirudin was PEGylated at His51, which showed that the acidic PEGylation operation prevented the PEGylation of active center (Lys47) of r-hirudin in principle. Mono-PEGylated product with purity higher than 95% was obtained as the pre-dominant product, and 34% of the anticoagulant activity was retained in vitro. The staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was improved to obtain perfect electrophoretic pattern in less than 5min. More accurate molecular weight was deduced due to the use of PEGs as molecular weight standards.

Key words: r-hirudin, histidine residues, SC-mPEG, mono-PEGylation, pH, anticoagulant activity

侯蓓蓓, 李世荣, 李晓晖, 修志龙. 单修饰重组水蛭素的设计, 制备及体外生物活性测定[J]. CIESC Journal.

HOU Beibei, LI Shirong, LI Xiaohui, XIU Zhilong. Design, preparation and in vitro bioactivity of mono-PEGylated recombinant hirudin[J]. .

参考文献

1 Rydel, T.J., Ravichandran, K.G., Tulinsky, A., Bode, W., Huber, R., Roitsch, C., Fenton, J.W., “The structure of a complex of recombinant hirudin and human α-thrombin”, Science, 249, 277—280(1990).
2 Rydel, T.J., Tulinsky, A., Bode, W., Huber, R., “Refined structure of the hirudin-thrombin complex”, J. Mol. Biol., 221, 583—601(1991).
3 Markwardt, F., “Past, present and future of hirudin”, Haemostasis, 21(Suppl 1), 11—26(1991).
4 Breddin, H.K., “Current developments in antithrombotic therapy: The role of antithrombin agents”, Pathophysiol. Haemost. Thromb., 32(Suppl 3), 1—8(2002).
5 Nowak, G., Markwardt, F., “Hirudin in desseminated in-travascular coagulation”, Haemostasis, 21, 142—148(1991).
6 Avgerinos, G.C., Turner, B.G., Gorelick, K.J., Papendieck, A., Weydemann, U., Gellissen, G., “Production and clinical development of a Hansenula polymorpha-derived PEGylated hirudin”, Semin. Thromb. Hemost., 27(4), 357—372(2001).
7 Markwardt, F., “The development of hirudin as an anti-thrombotic drug”, Thromb. Res., 74(1), 1—23(1994).
8 Wathion, N., “EMEA public statement on refludan (le-pirudin)”, http://www.emea.eu.int/pdfs/human/press/pus/ 2771702en.pdf(2002).
9 Roberts, M.J., Bentley, M.D., Harris, J.M., “Chemistry for peptide and protein PEGylation”, Adv. Drug. Deliv. Rev., 54, 459—476(2002).
10 Veronese, F., “Peptide and protein PEGylation: A review of problems and solutions”, Biomaterials, 22, 405—417(2001).
11 Yu, A.P., Jiang, Z.H., Zhong, G.S., Donh, C.N., Wu, Z.Z., “Isolation of PEGylated hirudin and analysis on its activity”, Pharm. Biotechnol., 11(5), 302—305(2004). (Chinese)
12 Bossavy, J.P., Sakariassen, K.S., Rubsamen, K., Thala-mas, C., Boneu, B., Cadroy, Y., “Comparison of the an-tithrombotic effect of PEG-hirudin and heparin in a hu-man ex vivo modelof arterial thrombosis”, Arterioscler. Thromb. Vasc. Bio., l19,1348—1353(1999).
13 Ulbricht, K., Bucha, E., Poschel, K.A., Stein, G., Wolf, G., Nowak, G., “The use of PEG-hirudin in chronic hemodi-alysis monitored by the ecarin clotting time: Influence on clotting of the extracorporeal system and hemostatic pa-rameters”, Clin. Nephrol., 65(3),180—190(2006).
14 Alexander, B., Burnand, K.G., Lattimer, C.L., Humphries, J., Gaffney, P.J., Eastham, D., Smith, A., “The effect of anticoagulation with subcutaneously de-livered polyethylene glycol conjugated hirudin and re-combinant tissue plasminogen activator on recurrent stenosis in the rabbit double-balloon injury model”, Thromb. Res., 113(2), 155—161(2004).
15 Poschel, K.A., Bucha, E., Esslinger, H.U., Ulbricht, K., Nortersheuser, P., Stein, G., Nowak, G., “Anticoagulant efficacy of PEG-hirudin in patients on maintenance hemodialysis”, Kidney Int., 65(2), 666—674(2004).
16 Laemmli, U.K., “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”, Nature, 227, 680—685(1970).
17 Manfred, M.K., “Detection and molecular weight deter-mination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis”, Anal. Biochem., 200, 244—248(1992).
18 Markwarkt, F., “Hirudin as inhibitor of thrombin”, Methods in Enzymology, 1st ed., Academic Press, New York(1957).
19 Melchior, W.B., Fahrney, D., “Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with α-chymotrypsin, pepsin,and pancreatic ribonuclease at pH 4”, Biochemistry, 9, 251—258(1970).
20 Lundblad, R.L., Noyes, C.M., Chemical Reagents for Protein Modification, CRC Press, Boca Raton (1984).
21 Miles, E.W., “Modification of histidyl residues in pro-teins by diethylpyrocarbonate”, Meth. Enzymol., 47, 431—442(1977).
22 Wylie, D.C., Voloch, M., Lee, S., Liu, Y.H., Can-non-Carlson, S., Cutler, C., Pramanik, B., “Carboxyalky-lated histidine is a pH-dependent product of PEGylation with SC-PEG”, Pharm. Res., 18(9), 1354—1360(2001).
23 Wong, S.S., Chemistry of Protein Conjugation and Crosslinking, CRC Press, Boca Raton (1991).

单修饰重组水蛭素的设计, 制备及体外生物活性测定

Design, preparation and in vitro bioactivity of mono-PEGylated recombinant hirudin

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