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| 重组别藻蓝蛋白(rAPC)的纯化与鉴定 |
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唐志红1,2,3, 李富超1,2, 吴少杰1,2, 葛保胜1,2, 林 凡1,2, 任育红1,2, 秦 松1
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1.中国科学院 海洋研究所;2.中国科学院 研究生院;3.烟台大学 海洋学院
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| 摘要: |
| 报道了重组大肠杆菌表达的别藻蓝蛋白的下游生产工艺研究,并对产物进行了分析鉴定。工程菌株P1先在NBS BioFlo3000型5L自动发酵罐进行高密度发酵,融合蛋白获得了高效表达,且主要在细胞内以可溶形式存在。纯化前先将获得的菌体超声破碎,然后经硫酸铵沉淀、疏水层析和离子交换层析三步纯化,接着对纯化的重组别藻蓝蛋白(rAPC)进行SDS- PAGE、PAGE、Westernblot等电点分析。证明已获得纯度为96.4%的rAPC,其等电点为6.0,并且具有与天然APC相似的抗原性。 |
| 关键词: 工程菌培养 重组别藻蓝蛋白(rAPC) 纯化 |
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| 基金项目:国家863计划资助项目(2001AA6204130);中国科学院知识创新项目(KZCX3–SW–215) |
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| Purification and characterization of recombinant allophycoc-yanin(rAPC) expressed in a recombinant Escherichia coli P1 |
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| Abstract: |
| This study reports a method to purify rAPC and analysis of the purified rAPC. The culture of the recombinant Escherichia coli strain was performed in a NBS BioFlo 3000 5 L fermentor(New Brunswick, USA); The final cell density was 16.8g dried cell/L broth and rAPC was produced as a water-soluble type. After sonication, the supernatant of free cell was fractionally precipited by ammonium sulfate. The protein was further purified by hydrophobic interaction chromatography and ion exchange chromatography. The purity of rAPC reached 96.4%; The purified rAPC was an acid protein(PI=6.0) and had a Mr value of approximate 60 ku; And the immunogenicity of rAPC is identical with that of its natural counterpart. |
| Key words: recombinant Allophycocyanin(rAPC) purification |