苯硼酸修飾之氧化鐵磁珠應用於蛋白質純化之研究

Abstract

近年來，文獻指出苯硼酸及其衍伸物可與特定具有順式雙醇基化合物產生良好的親和力並應用於生醫領域上，其常見的應用為在材料的表面修飾上苯硼酸進而可用來捕捉醣蛋白。此外，另有文獻指出苯硼酸與多巴胺 (Dopamine)、絲胺酸 (Serine)及組胺酸 (Histidine) 具有良好的親和力，其中與多巴胺有類似官能基側鏈的酪胺酸 (Tyrosine) 可用於蛋白質定量；絲胺酸可以增加蛋白質溶解度；連續六個組胺酸的胜肽 (His-tag) 被應用於蛋白質純化。如可使用具苯硼酸官能基的材料純化具有Tyr-tag、Ser-tag和/或His-tag的蛋白質，往後將可以應用此系統純化或固定化蛋白質。 本研究將苯硼酸修飾於氧化鐵磁珠表面並以傅立葉轉換紅外光譜 (FT-IR)、穿透式電子顯微鏡 (TEM)、茚三酮 (Ninhydrin) 試劑、比色分析、茜素紅S (Alizarin Red S, ARS) 試劑、進行表面鑑定，之後用於捕捉透過設計具有Tyr-tag、Ser-tag和/或His-tag的胜肽及綠色螢光蛋白 (GFP)，再利用其結構類似物，例如果糖和/或咪唑與上述胜肽或綠色螢光蛋白進行競爭反應，進一步探討磁珠捕捉能力及應用性。 結果顯示經過苯硼酸修飾後的磁珠可成功捕捉Tyr-tag、Ser-tag或His-tag的胜肽。進一步，其也可以有效地捕捉具有His-tag的綠色螢光蛋白，並可用200 mM 咪唑將綠色螢光蛋白沖提下來，可做為純化具有His-tag蛋白質的新方法。此外，同時具有His-tag及Ser-tag的綠色螢光蛋白可以用300 mM 果糖及 300 mM 咪唑混合溶液沖提下來，但是His-tag及Tyr-tag的綠色螢光蛋白不行，因此可以將具有His-tag及Tyr-tag的蛋白質應用於磁珠的固定化。 最後，本研究也成功地使用磁珠固定化自斷裂的蛋白質H12S6-GFP-(EAAAR)2-RFP，在pH 9的磷酸鹽緩衝液條件下讓RFP由磁珠上斷下來，未來可建構一鍋化的自斷裂純化系統。

The development of phenylboronic acid derivatives has attracted great interest in biomedical field recent years due to its good glycoprotein capture ability. Many researches indicated that phenylboronic acid and its derivatives could react with cis-diols forming boronate-ester complex which has been widely used in carbohydrate recognition. As reported in J. Chem. Biol by G. F. Whyte, phenylboronic acid also shows high binding affinity with dopamine, serine and histidine. The functional group of tyrosine is similar to dopamine which can be used in protein quantification; Serine can increase the protein solubility; His-tag can be used in protein purification. Utilize phenylboronic acid to capture proteins containing Tyr-tag, Ser-tag and/or His-tag is a new avenue of exciting fundamental. It may also be extended to protein purification and immobilization. In this study, we identified the surface of phenylboronic acid functionalized magnetic particles through Fourier transform infrared spectroscopy, transmission electron microscope, ninhydrin reagent, colorimetric analysis and ARS reagent. In order to test the capture ability of phenylboronic acid functionalized magnetic particles, we designed peptides and proteins both containing Tyr-tag, Ser-tag and/or His-tag. Then analogs such as fructose and/or imidazole was added to compete with green fluorescent protein (GFP). Our result proved that after modifying phenylboronic acid, magnetic particles could capture peptides containing Tyr-tag, Ser-tag or His-tag successfully. Further, it can also capture His-tag GFP very efficiently which could be easily collected by 200 mM imidazole elution buffer. Moreover, the GFP containing His-tag and Ser-tag can be eluted with the mixture solution, which is composed of 300 mM fructose and 300 mM imidazole, but the GFP containing His-tag and Tyr-tag can be not eluted by the above mixture solution. Therefore, the proteins containing His-tag and Tyr-tag can used to immobilize protein. This outcome may help improve the protein purification and immobilization in various bio-clinical applications. We also successfully immobilized auto-cleavable proteins, H12S6-GFP-(EAAR)2-RFP and red fluorescent protein (RFP), on magnetic particles which can be released under phosphate buffer at pH 9. This study establishes a robust one-pot auto-cleavable system that can be widely used in protein purification technology.