| 蛇毒类凝血酶calobin在毕赤酵母中的表达 |
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| 引用本文： | 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calobin在毕赤酵母中的表达[J].生物工程学报,2009,25(4):526-532. |
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| 作者姓名： | 袁盛凌 王芃 陶好霞 展德文 王艳春 王令春 刘纯杰 张兆山 |
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| 作者单位： | 中国人民解放军军事医学科学院,生物工程研究所,北京,100071 |
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| 摘 要： | 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 AccessionNo.U32937.1),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32kD的重组calobin蛋白,经甲醇诱导培养,表达产物可获得3.5g/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40kD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。
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| 关 键 词： | 蛇毒类凝血酶 毕赤酵母 calobin 纤维蛋白原 |
| 收稿时间： | 2008/8/14 0:00:00 |
Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris |
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| Shengling Yuan,Peng Wang,Haoxia Tao,Dewen Zhan,Yanchun Wang,Lingchun Wang,Chunjie Liu and Zhaoshan Zhang.Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris[J].Chinese Journal of Biotechnology,2009,25(4):526-532. |
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| Authors: | Shengling Yuan Peng Wang Haoxia Tao Dewen Zhan Yanchun Wang Lingchun Wang Chunjie Liu and Zhaoshan Zhang |
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| Affiliation: | Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China |
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| 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 a... |
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| Keywords: | thrombin-like enzyme Pichia pastoris calobin fibrinogen |
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