大肠杆菌表达的重组葡激酶-水蛭素融合蛋白的分离纯化及其二聚体分析

中国生物工程杂志

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大肠杆菌表达的重组葡激酶-水蛭素融合蛋白的分离纯化及其二聚体分析

钟根深于爱平靳继德蒋中华吴祖泽

军事医学科学院放射与辐射医学研究所军事医学科学院放射与辐射医学研究所军事医学科学院放射与辐射医学研究所军事医学科学院放射与辐射医学研究所军事医学科学院辐射与放射医学研究所

Purification of the Recombinant Fusion Protein Staphylokinase-Hirudin Expressed in Escherichia cloi and its Analysis of Self-association in Solution

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摘要： 采用阴离子交换和凝胶过滤色谱法纯化大肠杆菌高密度培养所表达的重组葡激酶－水蛭素融合蛋白（rSFH），经SDS-PAGE和RP-HPLC分析纯度达到98％以上，每升发酵液得率约0.7g。同时利用疏水色谱、MALDI-TOF对纯化过程中出现的rSFH同源二聚体的分析和表面疏水面积的计算及利用高效排阻色谱（HPSEC）分析NaCl、温度对rSFH的可逆二聚化行为的影响，认为疏水作用在rSFH的可逆二聚化行为中发挥着重要作用。

Abstract: The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from soluble E.coli lysate employing ion-exchange chromatographic (IEC) and gel filtration chromatographic (GFC) methods produced by high cell density cultivation strategy. This purification process resulted in greater than 98% pure product based on RP-HPLC and SDS-PAGE and yielded up to 0.7g per liter fermentation broth. The dimer and reversible self-association of rSFH presented in solution, after the average surface hydrophobicity(фsurface) analysis based on hydrophobic interaction chromatography (HIC) and mass spectrometry, are postulated to be the major contribution of hydrophobic effects, combining with the influences of salt(sodium chloride) and temperature on the monomer-dimer equilibrium based on size-exclusion high performance liquid chromatography (HPSEC). The demonstration here will be meaningful to other staphylokinase-base proteins of interest for thrombolytic therapy produced by genetic engineering in future.

收稿日期: 2006-10-24 出版日期: 2007-02-25

通讯作者: 吴祖泽

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引用本文:

钟根深,于爱平,靳继德,蒋中华,吴祖泽. 大肠杆菌表达的重组葡激酶-水蛭素融合蛋白的分离纯化及其二聚体分析[J]. 中国生物工程杂志, .

. Purification of the Recombinant Fusion Protein Staphylokinase-Hirudin Expressed in Escherichia cloi and its Analysis of Self-association in Solution. China Biotechnology, .

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I2/35

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