Efficient Expression, Purification and Functional Evaluation of Human Epidermal Growth Factor-Collagen Binding Domain Hybrid Protein on Growth Stimulation of Human Fibroblast Cells

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Efficient Expression, Purification and Functional Evaluation of Human Epidermal Growth Factor-Collagen Binding Domain Hybrid Protein on Growth Stimulation of Human Fibroblast Cells

机译：有效的表达式,净化和功能人类表皮生长的评价Factor-Collagen绑定域混合蛋白人类成纤维细胞的增长刺激

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Growth factors such as human epidermal growth factor (hEGF) have recently received high interest in regenerative medicine and pharmaceutical industries mainly due to their ability to restore tissues proliferation and improvement of their biological functions. In spite of various hEGF applications, its efficient expression in Escherichia coli could not yet reach an industrial reality mainly due to the lack of the ability of folding into the correct 3D structure because of three disulfide bonds in monomer hEGF. To address these challenges, here a fusion hEGF protein with a C terminus of collagen binding domain (CBD) along with intein protein with self-splicing property and ELP sequence was constructed by a three-step cloning procedure. This enabled us to purify recombinant hEGF without using chromatography columns. Following the confirmation of the construct by colony PCR, restriction enzymes analysis and sequencing, the 62 kDa band of ELP-INTEIN-hEGF-CBD were observed on SDS-PAGE and confirmed by western blotting. Subsequently, the mitotic activity in Balb/c 3T3 cells proliferation in presence of recombinant hEGF-CBD compared with commercial hEGF using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which showed that our purified recombinant protein stimulates the cell proliferation similar to the commercial protein. Our strategy could be considered as a new feasible approach to produce hEGF in E. coli for pharmaceutical and clinical applications.

机译：人类表皮生长增长等因素因子(高能)最近收到高再生医学和兴趣制药行业主要是由于他们恢复组织增殖和能力改善他们的生物功能。尽管各种高能应用程序,其效率表达在大肠杆菌可能还没有达到一个工业主要由于现实缺乏能力的折叠成正确的三维结构,因为三个二硫键单体高能。高能融合蛋白C末端的胶原蛋白绑定域名(CBD)连同intein蛋白质self-splicing财产和ELP序列由一个三步克隆过程。这使我们能够净化重组高能不使用色谱柱。确认菌落PCR的构造,限制性内切酶分析和排序,这些62 kDa群ELP-INTEIN-hEGF-CBD被观察到在sds - page和经免疫印迹。随后,在Balb / c 3 t3细胞有丝分裂活动细胞增殖在重组的存在hEGF-CBD与商业高能使用分析显示,我们的纯化重组蛋白质刺激细胞增殖相似商业的蛋白质。视为一种新的可行方法高能制药和临床的大肠杆菌应用程序。

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《Proceeding of the Singapore National Academy of Science》 |2019年第1期|35-45|共11页
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中图分类自然科学总论;
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Inteins; Escherichia coli; fusion proteins3T3 CellsPurificationfunctional evaluationgrowth stimulationGrowth FactorsDisulfide LinkagePharmaceutical IndustryCell ProliferationCloning;

机译：Inteins;大肠杆菌proteins3T3熔化CellsPurificationfunctional evaluationgrowthstimulationGrowth FactorsDisulfideLinkagePharmaceutical IndustryCell;

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Efficient Expression, Purification and Functional Evaluation of Human Epidermal Growth Factor-Collagen Binding Domain Hybrid Protein on Growth Stimulation of Human Fibroblast Cells

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