Gallus Sabrina, Peschke Theo, Paulsen Malte, Burgahn Teresa, Niemeyer Christof M, Rabe Kersten S
Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces 1 (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
Novartis Pharma AG Chemical and Analytical Development (CHAD), 4056, Basel, Switzerland.
Chembiochem. 2020 Aug 3;21(15):2126-2131. doi: 10.1002/cbic.202000102. Epub 2020 Apr 21.
The display of complex proteins on the surface of cells is of great importance for protein engineering and other fields of biotechnology. Herein, we describe a modular approach, in which the membrane anchor protein Lpp-OmpA and a protein of interest (passenger) are expressed independently as genetically fused SpyCatcher and SpyTag units and assembled in situ by post-translational coupling. Using fluorescent proteins, we first demonstrate that this strategy allows the construct to be installed on the surface of E. coli cells. The scope of our approach was then demonstrated by using three different functional enzymes, the stereoselective ketoreductase Gre2p, the homotetrameric glucose 1-dehydrogenase GDH, and the bulky heme- and diflavin-containing cytochrome P450 BM3 (BM3). In all cases, the SpyCatcher-SpyTag method enabled the generation of functional whole-cell biocatalysts, even for the bulky BM3, which could not be displayed by conventional fusion with Lpp-OmpA. Furthermore, by using a GDH variant carrying an internal SpyTag, the system could be used to display an enzyme with unmodified N- and C-termini.
在细胞表面展示复杂蛋白质对于蛋白质工程和生物技术的其他领域至关重要。在此，我们描述了一种模块化方法，其中膜锚定蛋白Lpp - OmpA和目标蛋白（乘客蛋白）分别作为基因融合的SpyCatcher和SpyTag单元独立表达，并通过翻译后偶联在原位组装。我们首先使用荧光蛋白证明了该策略能够将构建体安装在大肠杆菌细胞表面。然后，我们通过使用三种不同的功能酶——立体选择性酮还原酶Gre2p、同源四聚体葡萄糖1 - 脱氢酶GDH以及体积较大的含血红素和双黄素的细胞色素P450 BM3（BM3），展示了我们方法的适用范围。在所有情况下，SpyCatcher - SpyTag方法都能够生成功能性全细胞生物催化剂，即使对于体积较大的BM3也是如此，而BM3无法通过与Lpp - OmpA的传统融合进行展示。此外，通过使用携带内部SpyTag的GDH变体，该系统可用于展示N端和C端未修饰的酶。
