Computational and Synthetic Biology Laboratory, College of Life Sciences and Biotechnology, Korea University, Seoul, South Korea.
Appl Environ Microbiol. 2012 May;78(9):3051-8. doi: 10.1128/AEM.07004-11. Epub 2012 Feb 17.
Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 10(4) molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli.
自转运蛋白已被用作细胞表面展示的锚定支架，通过用待展示的异源蛋白替代其载体结构域。我们采用大肠杆菌的自转运蛋白（YfaL）作为细胞表面展示系统。为构建基于连接酶独立克隆（LIC）的展示系统，确定了 YfaL 中用于表面展示的关键区域。设计的系统对宿主细胞的生长或细胞表面上异源蛋白的过度表达没有不利影响。我们将单体红色荧光蛋白（mRFP1）作为报告蛋白进行功能性展示，并展示了来自 Saccharophagus degradans 2-40 的各种琼脂酶，包括 Aga86C 和 Aga86E，它们之前未能进行功能性表达。该系统可以展示大小从 25.3 到 143 kDa 的不同蛋白。我们还尝试通过在展示蛋白的 C 末端加入烟草蚀纹病毒蛋白酶切割位点来实现展示蛋白的受控释放。展示蛋白的最高水平为每个细胞 6.1×10^4 个分子，相当于生长活跃的大肠杆菌整个细胞表面的 5.6%。
