Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system
Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system
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DOI：
10.1016/j.pep.2008.05.009
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发表时间：
2008-09-01
影响因子：
1.6
通讯作者：
Miroshnikov, Anatoly I.
中科院分区：
文献类型：
--
作者：
Esipov, Roman S.;Stepanenko, Vasily N.;Miroshnikov, Anatoly I.
化学酶法合成人表皮生长因子（hEGF） cDNA，并将其克隆到表达载体pTWIN1 （New England Biolabs）中。在大肠杆菌中表达的重组融合蛋白由n端几丁质结合域、mini- interin Ssp dnaB域和c端hEGF多肽组成。在这个结构中，迷你链蛋白Ssp dnaB发挥了催化活性亚基的作用，能够在一定条件下进行自催化裂解，导致目标蛋白的分离。由于杂交蛋白的序列中含有几个半胱氨酸，其中一个在几丁质结合域，一个在mini-intein中，六个在hEGF中，因此有必要确定重组hEGF的最佳重折叠和纯化方案。在此基础上，提出了两种重组hEGF的纯化方案：第一种方案是将半胱氨酸还原的重组蛋白结合到几丁质柱上，将hEGF切割并洗脱，然后重新折叠形成合适的半胱氨酸桥。在第二种方案中，首先将整个杂交蛋白重新折叠形成二硫键，然后将其装载到亲和树脂上；重组hEGF在其天然状态下被分离和洗脱。尽管第一种方案更常见，适用于多种重组蛋白，但在重组hEGF的情况下，第二种方案被证明更具生产力和成本效益。(C) 2008爱思唯尔公司版权所有。
Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective. (C) 2008 Elsevier Inc. All rights reserved.