Lee Taek, Min Junhong, Hirakawa Hidehiko, Nagamune Teruyuki, Choi Jeong-Woo
Research Institute for Basic Science, Sogang University, Seoul, Republic of Korea; Department of Chemical and Biomolecular Engineering, Sogang University, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742, Republic of Korea.
School of Integrative Engineering, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Korea.
Colloids Surf B Biointerfaces. 2014 Aug 1;120:215-21. doi: 10.1016/j.colsurfb.2014.03.034. Epub 2014 May 21.
Recently, the fabrication of protein bilayer has been required for the development of protein or enzyme complex formation. In the present study, we fabricated a fusion protein bilayer composed of recombinant azurin-cytochrome P450, which was synthesized by a site-specific sortase-mediated ligation method. The Pseudomonas aeruginosa azurin was modified by DNA recombinant technique, for enzymatic ligation and immobilization. The Pseudomonas putida cytochrome P450 was also modified for enzymatic ligation. The recombinant metalloproteins were conjugated via the sortase A. The conjugation was confirmed by SDS-PAGE and UV-vis. Then, the prepared fusion protein was immobilized on Au substrate, by the self-assembly method. The Azu-P450 (recombinant azurin-cytochrome P450) fusion protein layer was confirmed by AFM (Atomic Force Microscopy) and SERS (Surface-enhanced Raman Spectroscopy), to confirm the fusion protein bilayer orientation. Moreover, the electrochemical property of Azu-P450 was observed by cyclic voltammetry (CV). As a result, the Azu-P450 fusion protein bilayer shows good orientation on the Au substrate. Also, the original redox property of this fusion protein bilayer has been well maintained. The proposed fusion protein bilayer can.
最近，蛋白质双层的制备对于蛋白质或酶复合物的形成发展是必需的。在本研究中，我们制备了一种由重组天青蛋白 - 细胞色素P450组成的融合蛋白双层，其通过位点特异性分选酶介导的连接方法合成。铜绿假单胞菌天青蛋白通过DNA重组技术进行修饰，用于酶促连接和固定。恶臭假单胞菌细胞色素P450也进行了修饰以用于酶促连接。重组金属蛋白通过分选酶A进行缀合。通过SDS - PAGE和紫外可见光谱确认了缀合。然后，通过自组装方法将制备的融合蛋白固定在金基底上。通过原子力显微镜（AFM）和表面增强拉曼光谱（SERS）确认了Azu - P450（重组天青蛋白 - 细胞色素P450）融合蛋白层，以确认融合蛋白双层的取向。此外，通过循环伏安法（CV）观察了Azu - P450的电化学性质。结果，Azu - P450融合蛋白双层在金基底上显示出良好的取向。而且，这种融合蛋白双层的原始氧化还原性质得到了很好的保持。所提出的融合蛋白双层可以。
