Design, expression, and renaturation of a lesion-targeted recombinant epidermal growth factor-von Willebrand factor fusion protein: efficacy in an animal model of experimental colitis.
Design, expression, and renaturation of a lesion-targeted recombinant epidermal growth factor-von Willebrand factor fusion protein: efficacy in an animal model of experimental colitis.
复制标题
针对病变的重组表皮生长因子-血管性血友病因子融合蛋白的设计、表达和复性：在实验性结肠炎动物模型中的功效。
DOI：
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发表时间：
2000
影响因子：
5.4
通讯作者：
E. Gordon
中科院分区：
文献类型：
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作者：
F. Hall;A. Kaiser;Ling Liu;Z. H. Chen;J. Hu;M. Nimni;R. Beart;E. Gordon
在本研究中，成熟的表皮生长因子（EGF）蛋白被工程化，以纳入一个高亲和力的胶原蛋白结合域（CBD）来自凝血血管性血友病因子，专门针对EGF的结肠病变。在大肠杆菌中表达了融合蛋白。大肠杆菌表达系统，通过金属螯合层析纯化，并通过氧化重折叠复性为可溶性生物活性生长因子。EGF-CBD融合蛋白在天然非靶向EGF被洗掉的条件下与胶原基质紧密结合。在生物测定中，EGF-CBD融合蛋白刺激NIH3T3细胞增殖，具有接近野生型的生物活性。体内结合研究表明，胶原蛋白靶向EGF，但不是非靶向EGF，积累在暴露的胶原蛋白在发炎的结肠腔表面的区域。最后，与非靶向EGF（隐窝数量= 52.2 +/-29.8; p = 0.027）和PBS对照（隐窝数量= 24）相比，单次结肠滴注胶原靶向EGF诱导了治疗后24小时肠隐窝更快速的再生（隐窝数量= 89.2 +/-8.1）。0 +/-22.9; p = 0.001）。总之，这些发现表明，结肠内递送胶原靶向EGF代表了急性或慢性炎症性肠病的潜在有效治疗策略。
In the present study, the mature epidermal growth factor (EGF) protein was engineered to incorporate a high affinity collagen-binding domain (CBD) derived from co-agulation von Willebrand factor, to specifically target EGF to colonic lesions. The fusion protein was expressed in an E. coli bacterial expression system, purified by metal chelate chromatography, and renatured by oxidative refolding into a soluble biologically active growth factor. The EGF-CBD fusion protein bound tightly to collagen matrices under conditions in which native non-targeted EGF was washed away. In biologic assays, the EGF-CBD fusion protein stimulated NIH3T3 cell proliferation with near wild-type biological activity. In vivo binding studies showed that the collagen-targeted EGF, but not the non-targeted EGF, accumulated at areas of exposed collagen on the luminal surface of the inflamed colon. Finally, a single colonic instillation of the collagen-targeted EGF-induced a more rapid regeneration of intestinal crypts 24 h after treatment (no. of crypts = 89.2+/-8.1) compared to the non-targeted EGF (no. of crypts = 52.2+/-29.8; p=0.027), and the PBS control (no. of crypts = 24. 0+/-22.9; p=0.001). Taken together, these findings indicate that intracolonic delivery of collagen-targeted EGF represents a potentially effective therapeutic strategy for acute or chronic inflammatory bowel disease.