Abstract
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 bacterial 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.
Keywords: Human epidermal growth factor, SUMO fusion, protein expression, Escherichia coli
Protein & Peptide Letters
Title: High-Level Expression and Purification of Human Epidermal Growth Factor with SUMO Fusion in Escherichia coli
Volume: 13 Issue: 8
Author(s): Zhijian Su, Yadong Huang, Quannan Zhou, Zhiling Wu, Xiaoping Wu, Qing Zheng, Changcai Ding and Xiaokun Li
Affiliation:
Keywords: Human epidermal growth factor, SUMO fusion, protein expression, Escherichia coli
Abstract: 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 bacterial 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.
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Cite this article as:
Su Zhijian, Huang Yadong, Zhou Quannan, Wu Zhiling, Wu Xiaoping, Zheng Qing, Ding Changcai and Li Xiaokun, High-Level Expression and Purification of Human Epidermal Growth Factor with SUMO Fusion in Escherichia coli, Protein & Peptide Letters 2006; 13 (8) . https://dx.doi.org/10.2174/092986606777841280
DOI https://dx.doi.org/10.2174/092986606777841280 |
Print ISSN 0929-8665 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5305 |
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