Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
Department I Internal Medicine, Medical Faculty, University of Cologne, Cologne, Germany.
Sci Rep. 2019 Jun 10;9(1):8410. doi: 10.1038/s41598-019-44883-z.
The demand for recombinant proteins for analytic and therapeutic purposes is increasing; however, most currently used bacterial production systems accumulate the recombinant proteins in the intracellular space, which requires denaturating procedures for harvesting and functional testing. We here present a novel FimH-based expression system that enables display of fully functional eukaryotic proteins while preventing technical difficulties in translocating, folding, stabilizing and isolating the displayed proteins. As examples, Gaussia Luciferase (GLuc), epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and epiregulin (EPRG) were expressed as FimH fusion proteins on the surface of E. coli bacteria. The fusion proteins were functionally active and could be released from the bacterial surface by specific proteolytic cleavage into the culture supernatant allowing harvesting of the produced proteins. EGFR ligands, produced as FimH fusion proteins and released by proteolytic cleavage, bound to the EGF receptor (EGFR) on cancer cells inducing EGFR phosphorylation. In another application of the technology, GLuc-FimH expressed on the surface of bacteria was used to track tumor-infiltrating bacteria by bioluminescence imaging upon application to mice, thereby visualizing the colonization of transplanted tumors. The examples indicate that the FimH-fusion protein technology can be used in various applications that require functionally active proteins to be displayed on bacterial surfaces or released into the culture supernatant.
对用于分析和治疗目的的重组蛋白的需求正在增加；然而，大多数当前使用的细菌生产系统将重组蛋白积累在细胞内空间中，这需要进行变性处理来收获和功能测试。我们在这里介绍了一种新颖的 FimH 表达系统，该系统能够展示具有完全功能的真核蛋白，同时防止在易位、折叠、稳定和分离展示蛋白方面出现技术困难。作为示例，Gaussia 荧光素酶 (GLuc)、表皮生长因子 (EGF)、转化生长因子-α (TGF-α) 和表皮调节素 (EPRG) 作为 FimH 融合蛋白在大肠杆菌表面表达。融合蛋白具有功能性，并且可以通过特异性蛋白水解切割释放到细菌表面进入培养上清液中，从而收获产生的蛋白质。作为 FimH 融合蛋白产生并通过蛋白水解切割释放的 EGFR 配体与癌细胞上的表皮生长因子受体 (EGFR) 结合，诱导 EGFR 磷酸化。在该技术的另一个应用中，细菌表面表达的 GLuc-FimH 在应用于小鼠后通过生物发光成像用于跟踪肿瘤浸润细菌，从而可视化移植肿瘤的定植。这些例子表明，FimH-融合蛋白技术可用于需要功能性蛋白在细菌表面展示或释放到培养上清液中的各种应用。
