Ohshima Tatsuki, Sakono Masafumi
Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama , 3190 Gofuku, Toyama 930-8555, Japan.
Bioconjug Chem. 2017 Nov 15;28(11):2687-2691. doi: 10.1021/acs.bioconjchem.7b00479. Epub 2017 Nov 3.
We produced a functional polymer whose framework comprised transthyretin (TTR) amyloid fibrils. In order to immobilize functional molecules onto the amyloid fibrils, transpeptidase sortase A (srtA), which catalyzes the covalent binding of LPXTG with polyglycine, was employed. After the preparation of the amyloid fibril of LPETGG-tagged TTR, immobilization of Gly5-fused GFP on the amyloid fibrils by srtA-mediated transpeptidation was carried out. SrtA recognized the amyloid fibrils consisting of an LPETGG-tagged TTR variant (L55P) as a good substrate, resulting in successful preparation of a GFP-immobilized amyloid. Intriguingly, the replacement of GFP with Gly5-fused luciferase was confirmed when the GFP-immobilized amyloids were mixed with Gly5-luciferase in the presence of srtA. Thus, it was found that functional molecules covalently immobilized on amyloid could be detached and substituted with other tagged molecules by using srtA.
我们制备了一种功能性聚合物，其骨架由转甲状腺素蛋白（TTR）淀粉样纤维组成。为了将功能分子固定在淀粉样纤维上，使用了催化LPXTG与聚甘氨酸共价结合的转肽酶分选酶A（srtA）。在制备LPETGG标记的TTR淀粉样纤维后，通过srtA介导的转肽作用将Gly5融合的绿色荧光蛋白（GFP）固定在淀粉样纤维上。SrtA将由LPETGG标记的TTR变体（L55P）组成的淀粉样纤维识别为良好的底物，从而成功制备了固定有GFP的淀粉样蛋白。有趣的是，当在srtA存在的情况下将固定有GFP的淀粉样蛋白与Gly5荧光素酶混合时，证实了Gly5融合的荧光素酶取代了GFP。因此，发现通过使用srtA，共价固定在淀粉样蛋白上的功能分子可以被分离并用其他标记分子替代。
