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1 生物工程学报 Chin J Biotech 2009, April 25; 25(4): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 工业生物技术 calobin 袁盛凌, 王芃, 陶好霞, 展德文, 王艳春, 王令春, 刘纯杰, 张兆山, : 蛇毒类凝血酶是临床上防治血栓栓塞性疾病的有效药物参照朝鲜蝮蛇 (Agkistrodon caliginosus, Korean Viper) 类凝血酶 calobin 基因序列 (GenBank Accession No. U ), 将人工合成的 calobin 基因克隆到酵母表达载体 ppiczαa, 于毕赤酵母中表达, 得到了分子量约为 32 kd 的重组 calobin 蛋白, 经甲醇诱导培养, 表达产物可获得 3.5 g/l 的高表达量重组蛋白经过阴离子交换柱 Q-Sepharose Fast Flow 和分子筛 Sephacryl-S-100 凝胶过滤层析等纯化步骤进行了初步纯化纯化后的重组 calobin 可以在纤维蛋白原平板上形成水解圈, 经 SDS-PAGE 实验显示, 重组蛋白能水解纤维蛋白原的 Aα 链, 产生一条约 40 kd 左右的降解带在实验中未能发现重组 calobin 对纤维蛋白原的凝固作用 : 蛇毒类凝血酶, 毕赤酵母, calobin, 纤维蛋白原 Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris Shengling Yuan, Peng Wang, Haoxia Tao, Dewen Zhan, Yanchun Wang, Lingchun Wang, Chunjie Liu, and Zhaoshan Zhang Institute of Biotechnology, Academy of Military Medical Sciences, Beijing , China Abstract: Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/l in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kd shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aα-chain of fibrinogen specifically, and produced an appropriately 40 kd new band. However, we failed to find its fibrin-clot formation activity. Keywords: thrombin-like enzyme, Pichia pastoris, calobin, fibrinogen,, [1], (Thrombin-like enzyme),,, A(Fibrinopeptide A, FPA) B(Fibrinopeptide Received: August 14, 2008; Accepted: February 13, 2009 Corresponding author: Chunjie Liu. Tel/Fax: ; Liucj@nic.bmi.ac.cn

2 : calobin 527 B, FPB),,, XⅢ,,,,,,,, [2,3],,, Yang gussurin gloshedobin Pichia pastoris [4,5], 2 You [6] [7],,, 200 NIH (Agkistrodon caliginosus, Korean Viper) calobin 1996, (226 NIH) [8] Aα, Bβ calobin [9], calobin, calobin Aα, 1 材料与方法 1.1 材料 JM109, GS115 ppiczαa Taq Pyrobest TaKaRa ( / ) Difco, D- Japan,, YPD BMGY BMMY, Invitrogen EasySelect TM Pichia Expression Kit (From bovine, 52 U/mg) (From bovine) Sigma, ussurin (10 U/ ) 1.2 方法 ppiczαa-calobin (Y-F): 5 -CCGCTCGAGAAAAGAGT CATTGGTGGTGATGAATG-3, Xho I (Y-R): 5 -GCTCTAGAACTCACGGCG GGCAGGACGCATCAG -3, Xba I calobin pet22b(+)-calobin [10],,, : 94 o C 5 min; 94 o C 35 s, 59 o C 40 s, 72 o C 60 s, 25 ; 72 o C 10 min PCR, Xho I Xba I,, ppiczαa, JM109, 25 μg/ml Zeocin LB, 16 h, Promega DNA Purification System : Invitrogen EasySelect TM Pichia Expression Kit ppiczαa-calobin Bgl II Sac I,, 1 μg/μl, 80 μl 5 μg DNA,, 1500 V 25 μf 100 ohms 4.2 s, 1 ml 1.0 mol/l, 30 o C, 1 2 h, 200 μl, YPDS, 29 o C 36 h PCR Invitrogen EasySelect TM Pichia Expression Kit DNA 2 : α-factor 5 -GAAGC TGTCATCGGTTACTCAGAT-3 3 AOX1 primer ; Y-F Y-R PCR : 94 o C 5 min; 94 o C 60 s, 55 o C 45 s, 72 o C 75 s, 30 ; 72 o C 10 min PCR, 3 AOX1 primer

3 528 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4, Invitrogen EasySelect TM Pichia Expression Kit h 1 ml, 0.5% r/min 1 min, 10 μl, SDS-PAGE o C 1500 g 10 min,, 70%, 4 o C, 6000 r/min 4 o C 10 min, 1/ mol/l Tris Cl (ph 7.45), 24 h, 12 h 1 Q-sepharose Fast Flow (2.0 cm 20 cm), 20 ml, 0.02 mol/l Tris Cl (ph 7.45) 5, 20, NaCl 0~1.0 mol/l,,, Q-sepharose Fast Flow, NaCl : 2 NaCl mol/l, 20 NaCl mol/l, 3 NaCl mol/l, 0.5 ml, 0.05 mol/l Tris Cl (ph 7.45) Sephacryl S-100(1.6 cm 90 cm),,, SDS-PAGE ) [11] : 50 mmol/l Tris Cl (ph 7.45) +1%,, 45 o C, 10 ml D=90 mm 50 mmol/l Tris Cl (ph 7.45) +2%,, 45 o C ; 0.8%, 45 o C 2, 10 ml,,, 37 o C, 2) : 50 mmol/l Tris Cl (ph 7.45) 0.4%, 100 μl, 37 o C, 3) SDS-PAGE : : 0.5% [50 mmol/l Tris Cl (ph 7.45) ], : 1 calobin: 380 μg/ml; 2 : 0.4 U/mL; 3ussurin: 0.4 U/mL ml, 25 μl 25 μl,,, 2 SDS, 20 o C 25 μl 25 μl 50 mmol/l Tris Cl(pH 7.45) 100 o C 3 min, r/min 2 min, 6 μl 2 结果 2.1 ppiczαa-calobin 载体的构建 calobin, Xho I Xba I, α-factor Kex2 calobin PCR 1A, 720 bp PCR, Xho I Xba I, ppiczαa JM109, 25 mg/l Zeocin LB, ( 1B), Bgl II, GS 酵母染色体的重组和 PCR 鉴定 36 h, 0.5~0.7 mm 1000 μg/ml Zeocin YPDS,, DNA, 2 α-factor 3 AOX1 primer,, PCR 1000 bp ; calobin Y-F Y-R, PCR 720 bp PCR ( 2), lane3 lane4, lane3, 3 AOX1 primer ( ),, PCR calobin,

: calobin 529 图 1 质粒 ppiczαa-calobin 的构建和鉴定 Fig. 1 Construction and identification of plasmid ppiczαa-calobin.

1: plasmid ppiczαa digested with Xho I; 2: plasmid ppiczαa-calobin digested with Xho I and Xba I. (C) the plasmid ppiczαa-calobin was linearized with restriction enzyme Bgl II or Sac I.

1: PCR products from chromosome DNA of GS115 by a pair of primers α-factor and 3 AOX1 primer; 2: PCR products from chromosome DNA of GS115 by a pair of primers Y-F and Y-R; 3: PCR products from

4 : calobin 529 图 1 质粒 ppiczαa-calobin 的构建和鉴定 Fig. 1 Construction and identification of plasmid ppiczαa-calobin. (A) PCR products representing calobin gene was amplified from plasmid pet22b(+)-calobin [10]. (B) New plasmid ppiczαa-calobin was identificated with restriction enzymes. 1: plasmid ppiczαa digested with Xho I; 2: plasmid ppiczαa-calobin digested with Xho I and Xba I. (C) the plasmid ppiczαa-calobin was linearized with restriction enzyme Bgl II or Sac I. 2,,, 3, 32 kd, 43% Folin 8.1 g/l( ), 3.5 g/l 图 2 采用 PCR 方法对 GS115 阳性整合子进行鉴定 Fig. 2 Confirmation of ppiczαa-calobin recombination into chromosome DNA of GS115 by PCR. 1: PCR products from chromosome DNA of GS115 by a pair of primers α-factor and 3 AOX1 primer; 2: PCR products from chromosome DNA of GS115 by a pair of primers Y-F and Y-R; 3: PCR products from chromosome DNA of positive colony by α-factor and 3 AOX1 primer; 4: PCR products from chromosome DNA of positive colony by primers Y-F and Y-R; M: DNA marker. 2.3 ppiczαa-calobin 的诱导培养, BMGY, OD 600 =2, Invitrogen EasySelect TM Pichia Expression Kit 48 h,, SDS, SDS-PAGE 6, 图 3 ppiczαa-calobin 的诱导表达产物 (48 h) Fig. 3 Expression products of ppiczαa-calobin induced for 48 h., 4, 24 h,,

530 ISSN1000-3061 CN11-1998/Q Chin J Biotech April 25, 2009 Vol.25 No.4,, 7 d (168 h),, 36 48 h 2.

4 Effects of harvest time on the accumulation of recombinant calobin.

5 530 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4,, 7 d (168 h),, h 2.5 重组 calobin 的活性测定 ( ): 15 μg calobin, thrombin ussurin, 37 o C 10 h, calobin, ( 6), ussurin calobin 图 4 ppiczαa-calobin 诱导不同时间后表达产物的积累 Fig. 4 Effects of harvest time on the accumulation of recombinant calobin. M: protein marker; 1: GS115 induced for 96 h; 2~9: GS115/pPICZαA-calobin induced for 3, 12, 24, 48, 72, 96, 120, 168 h, respectively. 2.4 GS115/pPICZαA-calobin 诱导表达产物的纯化 48 h 4 o C, 70%,, Q-sepharose Fast Flow (2.0 cm 20 cm), mol/l NaCl,, SDS-PAGE,,,, NaCl,, 0.5 ml, Sephacryl S-100(1.6 cm 90 cm),, SDS-PAGE, 5 图 5 重组 calobin 的纯化 Fig. 5 Purification of recombinant calobin. M: protein marker; 1: purified calobin; 2: fermentation supernatant of GS115/ ppiczαa-calobin. 图 6 重组的类凝血酶 calobin 对纤维蛋白原的作用 Fig. 6 Incubation of recombinant thrombin-like enzyme calobin with fibrinogen. 1: recombinant calobin 15 μg; 2: thrombin (bovine) 0.25 U; 3: ussurin 0.25 U; 4: negative control. calobin, 1 h,,, ussurin calobin ussurin SDS-PAGE ( 7) Aα Bβ γ, 3 ( 7A) calobin Aα, 20 min, 40 kd ( 7B) ussurin Aα, 7C Aα, Aα Bβ

6 : calobin 531 A- [2] calobin A-,,,, calobin, Ser,, C [12] Ser,,,,,,, (Crotalus adamanteus) Crotalase, [13] 图 7 牛凝血酶重组 calobin 和天然 ussurin 在不同时间点对纤维蛋白原的水解状况 Fig. 7 Incubation of fibrinogen with different enzymes at different time intervals. (A) Fibrinogen was incubated with bovine thrombin (sigma) and aliquots were taken for SDS- PAGE at different time intervals. M: protein marker; 2 9: 0, 1.5, 4, 10, 20, 30, 60, and 120 min. (B) Fibrinogen was incubated with recombinant calobin for 0, 20, 40, 60, 90, 120, 180 and 240 min (2 9). (C) Fibrinogen in the presence of native ussurin was for, 0, 3, 7, 15, 30, 45 and 100 min (2 8). 1: Fibrinogen in the absence of enzyme was used as control. 4 讨论 gussurin gloshedobin Pichia pastoris,, [4,5] You [6] [7],,, 200 NIH, calobin,, calobin,, Aα, calobin,,, calobin (Agkistrodon caliginosus),, calobin calobin2 CPI enzyme-1 CPI enzyme-2 Kininogenase-1 Kininogenase-2 Shimokawa [14] 4, 4, CPI enzyme-2 A- B-, Kininogenase-2 A-, Bβ Hahn [15] CPI enzyme-2, calobin : 1) 2, 258, 262 ; 2), 18+6; 3), His41 Asp86 Ser180, 2, His43 Asp88 Ser182; 4) DNA : : 98%; : 84%, CPI enzyme-2 calobin DNA, Ser,,,,,

7 532 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4 Ser,, Aα, REFERENCES [1] Guan LF, Qi ZW. The research on the Agkistrodon halys Pallas snake venom thrombin-like enzyme. Acta Biochem Biophys Sin, 1982, 14(4): ,. (Agkistrodon halys Pallas)., 1982, 14(4): [2] Tan GP. Chinese Snake Venom. 2nd Ed. Nanning: Guangxi Science and Technology Press, :, [3] Lathan LO, Staggers SL. Ancrod: The use of snake venom in the treatment of patients with heparin-induced thrombocytopenia and thrombosis undergoing coronary artery bypass grafting: Nursing management. Heart Lung, 1996, 25(6): [4] Yang Q, Hu XJ, Xu XM, et al. Cloning, expression and purification of Gussurobin, a thrombin-like enzyme from the snake venom of Gloydius ussuriensis. Acta Bioch Bioph Sin, 2002, 34(1): 6 10.,,,. Gussurobin., 2002, 34(1): [5] Yang Q, Hu XJ, Xu XM, et al. Expression, purification and partial characterization of recombinant Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis. Prog Biochem Biophys, 2002, 29(3): [6] You WK, Choi WS, Koh YS, et al. Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris. FEBS Lett, 2004, 571(1-3): [7] Li ZF, Yu XL, Huang JL, et al. Recombinant batroxobin expressed highly in Pichia pastoris. Chin J Biotech, 2007, 23(3): ,,,.., 2007, 23(3): [8] Hahn BS, Yang KY, Park EM, et al. Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean Viper). J Biochem(Tokyo), 1996, 119(5): [9] Yuan SL, Duan HQ, Zhang ZS. The design, chemical synthesis and cloning of cdna of Agkistrodon caliginosus thrombin-like enzyme Calobin. Lett Biotechnol, 2003, 14(5): ,,. Calobin cdna., 2003, 14(5): [10] Yuan SL, Duan HQ, Liu CJ, et al. The role of thioredoxin and disulfide isomerase in the expression of the snake venom thrombin-like enzyme calobin in Escherichia coli BL21(DE3). Protein Expr Purif, 2004, 38(1): [11] Maeda M, Satoh S, Suzuki S, et al. Expression of cdna for batroxobin, a thrombin-like enzyme. J Biochem (Tokyo), 1991, 109(4): [12] Deshimaru M, Ogawa T, Nakashima K, et al. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett, 1996, 397(1): [13] Markland FS. Snake venoms and the hemostatic system. Toxicon, 1998, 36(12): [14] Shimokawa K, Takahashi H. Comparative study of fibrinogen degradation by four arginine ester hydrolases from the venom of Agkistrodon caliginosus(kankoku- Mamushi). Toxicon, 1995, 33(2): [15] Hahn BS, Beak K, Kim WS, et al. Molecular cloning of capillary permeability-increasing enzyme 2 from Agkistrodon caliginosus(korean viper). Toxicon, 1998, 36(12):

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