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Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation

文献类型： 外文期刊

作者： Wang, Yunpeng 1 ; Fan, Jieying 1 ; Ahamd, Niaz 2 ; Xin, Wen 4 ; Wei, Zhengyi 1 ; Xing, Shaochen 1 ;

作者机构： 1.Jilin Acad Agr Sci, Inst Agr Biotechnol, Changchun, Peoples R China

2.Natl Inst Biotechnol & Genet Engn NIBGE, Agr Biotechnol Div, Faisalabad, Pakistan

3.Pakistan Inst Engn & Appl Sci PIEAS, Dept Biotechnol, Islamabad, Pakistan

4.Beijing TransGen Biotech Co Ltd, Beijing, Peoples R China

5.Guangxi Acad Agr Sci, Maize Res Inst, Nanning, Peoples R China

6.Jilin Acad Agr Sci, Inst Agr Biotechnol, Changchun 130033, Peoples R China

7.Guangxi Acad Agr Sci, Maize Res Inst, Nanning 530007, Peoples R China

关键词： Chloroplast transformation; epidermal growth factor; biopharming; tobacco

期刊名称：GROWTH FACTORS （ 影响因子：2.394； 五年影响因子：2.765 ）

ISSN： 0897-7194

年卷期：

页码：

收录情况： SCI

摘要： Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 x His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene aadA, hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16-25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.

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