Production of Hepatopoietin Cn in High-Cell-Density Cultures of Recombinant Escherichia coli and Detection of its Antioxygen Activity
Production of Hepatopoietin Cn in High-Cell-Density Cultures of Recombinant Escherichia coli and Detection of its Antioxygen Activity
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重组大肠杆菌高密度培养肝生成素Cn的产生及其抗氧活性检测
DOI：
10.1007/s12033-010-9318-x
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发表时间：
2011-02
影响因子：
2.6
通讯作者：
中科院分区：
文献类型：
--
作者：
肝生成素Cn （Hepatopoietin Cn, HPPCn）是富含亮氨酸的酸性核蛋白（LANP）家族的一员，是一种新型的人肝脏生长因子，通过激活多种信号通路，在体内和体外刺激DNA合成和肝细胞增殖。我们进行了重组人HPPCn （rhHPPCn）蛋白的中试生产，以阐明其在肝脏保护中的作用。在40-l的生物反应器中培养含有pET-24a-HPPCn表达载体的大肠杆菌BL21 （DE3）菌株。利用该菌株进行高密度补料分批发酵，产生了以包涵体形式存在的高浓度rhHPPCn蛋白。在蛋白质洗涤和溶解后，通过稀释变性剂实现rhHPPCn的再折叠。然后，用层析纯化再生蛋白。所得蛋白的生物活性评价表明，rhHPPCn可以减轻乙醇诱导的肝细胞损伤的氧化应激。我们成功地大规模生产了具有生物活性的rhHPPCn蛋白，并通过上述的再生和纯化程序获得了每克干细胞重量大于40 mg rhHPPCn的产量。得到的体积产量相当于每升培养肉汤640毫克的生物活性rhHPPCn。此外，重组HPPCn蛋白被证实可以保护肝细胞免受乙醇诱导的氧化应激。
Hepatopoietin Cn (HPPCn), a member of the leucine-rich acidic nuclear protein (LANP) family, is a novel human hepatic growth factor that stimulates DNA synthesis and hepatocyte proliferation in vivo and in vitro by activating several signaling pathways. We performed pilot-scale production of recombinant human HPPCn (rhHPPCn) protein to elucidate its role in liver protection. An Escherichia coli BL21 (DE3) strain containing a pET-24a-HPPCn expression vector was cultivated in a 40-l bioreactor. High-cell-density fed-batch fermentation using this strain yielded a high concentration of rhHPPCn protein in the form of inclusion bodies. After washing and solubilization of the protein, rhHPPCn refolding was achieved by dilution of the denaturant. Then, the renatured protein was purified chromatographically. Assessment of the biological activity of the obtained protein showed that rhHPPCn could attenuate the oxidative stress in ethanol-induced hepatocyte injury. We successfully achieved large-scale production of biologically active rhHPPCn protein and obtained yields greater than 40 mg rhHPPCn per gram dry cell weight by applying the above-mentioned renaturation and purification procedures. The obtained volumetric yield corresponds to 640 mg of biologically active rhHPPCn per liter of culture broth. Additionally, the recombinant HPPCn protein was confirmed to protect hepatocytes against ethanol-induced oxidative stress.
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影响因子：
20.1
作者：
Tampo, Y;Kotamraju, S;Kalyanaraman, B
通讯作者：
Kalyanaraman, B
影响因子：
13.5
作者：
Cui, Chun-Ping;Zhang, Da-Jin;Wu, Chu-Tse
通讯作者：
Wu, Chu-Tse
影响因子：
4.1
作者：
RIESENBERG, D;SCHULZ, V;DECKWER, WD
通讯作者：
DECKWER, WD
影响因子：
2.8
作者：
Passarinha, L. A.;Bonifacio, M. J.;Queiroz, J. A.
通讯作者：
Queiroz, J. A.
影响因子：
2.8
作者：
Singh, SM;Panda, AK
通讯作者：
Panda, AK