Yoo Gu, Saenger Thorsten, Bong Ji-Hong, Jose Joachim, Kang Min-Jung, Pyun Jae-Chul
Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-ro, Seo-dae-mun-gu, Seoul 120-749, Republic of Korea.
Institute of Pharmaceutical and Medical Chemistry, University of Muenster, Muenster, Germany.
Biochim Biophys Acta. 2015 Dec;1848(12):3126-33. doi: 10.1016/j.bbamem.2015.09.018. Epub 2015 Sep 25.
In this work, two proteins, Z-domains and bovine casein, were auto-displayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different auto-display vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-auto-displayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-auto-displayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-auto-displayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-auto-displayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with auto-displayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.
在本研究中，通过共转化两种不同的自展示载体，将两种蛋白质——Z结构域和牛酪蛋白自动展示在同一大肠杆菌细胞的外膜上。基于SDS-PAGE光密度测定法，Z结构域和牛酪蛋白分别以3.12×10⁵和1.55×10⁵个蛋白质/大肠杆菌细胞的水平表达。共自动展示的Z结构域具有抗体结合活性，而牛酪蛋白具有黏附特性。对共自动展示蛋白质的大肠杆菌进行了荧光辅助细胞分选（FACS）分析。共自动展示Z结构域和牛酪蛋白的大肠杆菌由于疏水相互作用而聚集。为了应用于免疫测定，在（1）固定大肠杆菌和（2）形成外膜层之后评估Z结构域活性。固定在聚苯乙烯微孔板上的共自动展示两种蛋白质的大肠杆菌具有与仅自动展示Z结构域的大肠杆菌相同的抗体结合活性。通过抗体结合活性测量，共转化大肠杆菌的外膜层中Z结构域和牛酪蛋白的表达比例为1:2。
