藉由訊號放大偵測蛋白質之半合成蛋白探針

蛋白質為人體之重要組成，調控人體許多新陳代謝。蛋白質失調會造成許多疾病，因此偵測蛋白質成為重要課題。偵測蛋白質之技術被廣為開發，其中以酵素免疫分析法最廣為人知。但這些技術皆需繁瑣的前處理，我們希望發展一個半合成訊號放大探針偵測蛋白質。透過生物重組蛋白技術合成SNAP-hCAII，以SNAP-tag 標記上有機探針。此探針含有磺胺抑制劑結構(sulfonamide, SA)、O6-苄基鳥嘌呤(O6-benzylguanine，BG)及生物素(biotin)。磺胺抑制劑結構(sulfonamide, SA)可抑制人類碳酸酐II(hCAII)活性，形成封閉系統。加入抗生物素蛋白(avidin)後，產生立障效應(steric effect)，使磺胺抑制劑結構(SA)脫離，hCAII恢復活性，得以水解Fluorescein diacetaete，產生訊號放大的螢光效果。此系統抑制效果不佳，探討SNAP蛋白是否會影響hCAII之抑制效果，嘗試以hCAII和SNAP-hCAII進行螢光測試，發現使用hCAII比SNAP-hCAII有更高的螢光增益。因此之後發展捨棄合成O6-benzylguanine，且蛋白質使用hCAII，不必經過生物選殖技術合成SNAP-hCAII，此探針具有更佳的螢光增益及便利性。

關鍵字

訊號放大；蛋白質偵測

並列摘要

Protein plays an important role in our body. Enzyme is a kind of protein. It can regulate metabolism. Many disease is due to deregulatory of protein. Thus, detection of protein is an important issue. Nowadays, there are two famous detection methods. One is enzyme-linked immunosorbent assay (ELISA)which utilizes antibody to obtain specific binding for protein detection. The other is Western blot. It combines gel electrophoresis to separate protein and detects by antibody. This two methods are time consuming , high cost and require multi-steps operation. In this thesis, we described a new enzyme amplified method for protein detection based on semisynthetic protein sensor. Through molecular biology and organic synthesis, we constructed a recombinant protein SNAP-HCA and a synthetic compound. As a proof-of-principle, we will demonstrate the design of our semisynthetic protein sensor for the detection of avidin. The function of SNAP-tag protein is to facilitate specific labeling of the the synthetic compound to the recombinant protein, while the function of HCA is to regulate enzyme amplification during protein detection. The synthetic compound consists of three functional units and they are benzylguanine (BG) for specific labeling to the recombinant protein via SNAP-tag, biotin group for the binding and recognition with avidin, and a sulfonamide inhibitor for the regulation of HCA activity for signal amplification. In the absence of avidin, the protein sensor will exit in a closed conformation which will prevent the HCA enzyme to catalyze the hydrolysis of nonfluorescent FLDA to strongly fluorescent FL product. In the presence of avidin, the protein will bind to biotin and generate steric effects which should open up the protein sensor. Under this circumstance, the affinity of sulfonamide and HCA is weakened and the signal amplification can thus occur.

並列關鍵字

signal amplification ； protein detection

參考文獻

34. Li, J.; Fu, H. E.; Wu, L. J.; Zheng, A. X.; Chen, G. N.; Yang, H. H., Anal. Chem., 2012, 84, 5309−5315

35. Miranda, O. R.; Chen, H. T.; You, C. C.; Mortenson, D. E.; Yang, X. C.; Bunz , U. H. F.; Rotello, V. M., J. Am. Chem. Soc., 2010, 132, 5285–5289

29. Huang, Y.; Chen,J.; Zhao, S.; Shi, M.; Chen. Z. F.; Liang, H., Anal. Chem. 2013, 85, 4423−4430

1. Nelson DL, Cox MM, Lehninger's Principles of Biochemistry-W. H. Freeman and Company4th Ed, 2005.

15. (a)Zhu, L.; Anslyn, E. V., Angew. Chem. Int. Ed., 2006, 45, 1190-1196(b)Scrimin, P.; Prins, L. J., Chem. Soc. Rev., 2011, 40, 4488–4505

國際替代計量

藉由訊號放大偵測蛋白質之半合成蛋白探針

全文下載

主題瀏覽