High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli
High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli
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DOI：
10.2174/092986606777841280
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发表时间：
2006-01-01
影响因子：
1.6
通讯作者：
Li, Xiaokun
中科院分区：
文献类型：
--
作者：
Su, Zhijian;Huang, Yadong;Li, Xiaokun
人表皮生长因子(HEGF)可刺激多种细胞类型的分裂，具有潜在的临床应用价值。然而，活性hEGF在大肠杆菌中的高效表达并不成功，因为该蛋白含有三个分子内的二硫键，这些键在细菌的胞内环境中很难正确形成。为了解决这一问题，我们将hEGF基因与一个小泛素相关修饰基因(SUMO)融合在一起，合成了在Origami(DE3)菌株中高效表达的SUMO-hEGF融合基因。可溶性融合蛋白SUMO-hEGF的最适表达水平可达菌体总蛋白的38.9%。融合蛋白经Ni-NTA亲和层析纯化后，经相扑专一性酶切得到天然的hEGF，再经Ni-NTA亲和层析纯化。反相高效液相色谱检测结果表明，重组裂解的hEGF纯度大于98%。经N端氨基酸序列测定和MALDI-TOF质谱分析，证实了纯化的hEGF的一级结构。用四甲基偶氮唑法测定，纯化的hEGF对Balb/c 3T3细胞的促有丝分裂活性与商品化的hEGF相当。
Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacteria] intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.