title: High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor inEscherichia coli.
abstract: Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF inEscherichia colihas not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure usingin vivosystem. To tackle this problem, we fusedMxeGyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinantE. colisystem and could be a better strategy to produce native hEGF for pharmaceutical development.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Jieying
authors.initials: J
authors.lastname: Yu
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Jinglian
authors.initials: J
authors.lastname: Lin
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Shaomin
authors.initials: S
authors.lastname: Wu
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China; Guangdong Province Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Shan
authors.identifier: 0000-0002-5211-0872
authors.initials: S
authors.lastname: Li
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Jufang
authors.identifier: 0000-0002-9908-1001
authors.initials: J
authors.lastname: Wang
mesh_terms: D016475:3T3 Cells; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects*; D003001:Cloning, Molecular / Q000379:methods; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D025842:SUMO-1 Protein; D006639:Histidine; D004815:Epidermal Growth Factor; D027081:DNA Gyrase