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首页> 外文期刊>Protein Expression and Purification >Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli
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Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli

机译：重组人表皮生长因子包涵体的溶解和重折叠，使用大肠杆菌的扩展床吸附色谱法

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Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity >99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 x 10(5) IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results. (c) 2008 Elsevier Inc. All rights reserved.
机译：在各种内源性生长因子中，表皮生长因子（EGF）在皮肤，角膜和胃肠道等组织的正常伤口愈合中起着重要作用。各种研究已经证明，补充重组人EGF（rhEGF）可以显着增强伤口愈合。在目前的工作中，具有聚精氨酸序列的高水平表达系统被用于产生重组人EGF（rhEGF）作为包涵体。将包涵体溶解，并通过使用扩展床吸附色谱法将蛋白质重新折叠。用适当浓度的胰蛋白酶消化变性的蛋白质，然后通过离子交换色谱法（Toyopearl-SP）纯化消化的rhEGF，从而获得生物活性蛋白质。该方法是最短的纯化步骤，减少了纯化步骤，因此无需使用制备型反相HPLC（RP-HPLC）进行最终纯化，这是一项昂贵的技术。纯化的蛋白质通过RP-HPLC分析，显示纯度> 99％，尺寸排阻色谱图显示，聚集体最少，活性蛋白质变性为99％。与NIBSC标准（rDNA衍生的EGF的第一国际标准，Code 91/530）相比，纯化的rhEGF的比活性为5 x 10（5）IU / mg蛋白。该方法已成功地在100 L发酵规模上采用，基于rhEGF的制剂已商业化，商标名为REGEN D，具有出色的临床效果。 （c）2008 Elsevier Inc.保留所有权利。

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