發展位向專一蛋白質修飾及固化方法

Development of site-specific methods for protein modification and immobilization

李本元(Li, Ben Yuan)

指導教授 ： 林俊成

國立清華大學/理學院/化學系/博士(2016年)

摘要

蛋白質的化學修飾為化學生物學相關研究的重要工具。為了避免蛋白質活性的降低，蛋白質位向專一的修飾在近幾年中以被廣泛的探討。本論文主要是專注於蛋白質位向專一的修飾，並研究蛋白質透過氟標記以及DNA標記固化在固相載體之活性。
氟化學為近年快速發展的領域，其特性為氟分子之間有很強的作用力，此特殊的作用力可用來抑制蛋白質固化時所造成的非專一性吸附，因此於本篇論文中，我們發展了兩種位向專一修飾氟標記於蛋白質的策略；其中，目標蛋白－綠色螢光蛋白、麥芽糖結合蛋白以及麩胺基硫轉移酶利用 intein 蛋白質表現系統表達，之後透過自然化學鍵結反應，將含有磺基丙胺酸的氟探針修飾在蛋白質的 C 端。另一方面，anti-蓖麻毒素抗體則是透過硼酸可與二元醇反應形成硼酯的特性，將含有硼酸的氟探針修飾在抗體的醣體上。修飾氟探針的蛋白質，可透過簡單的混合，固化在固相載體上。氟-氟非共價鍵作用力相當穩定，除了可以承受不斷的清洗之外，亦可有效地抑制蛋白質的非專一性吸附。
由於 DNA 生物檢測技術具有快速且低成本的優勢，近年來有越來越多的科學家投入其研究當中。於本論文中，透過 DNA 鹼基對之間的作用力，固化蛋白質於磁性奈米粒子，並探討固化前後蛋白質的活性差異。目標酵素－磷酸葡萄糖胺胸苷轉移酶及半乳醣激酶可透過 2-氰基苯並噻唑與半胱胺酸進行縮和反應，專一地於蛋白質的 C 端建構 DNA 分子，除此之外，修飾 DNA之酵素可透過 DNA 互補對之間的作用力，固化在磁性奈米粒子；當高於解構溫度時，固化之酵素會從磁性奈米粒子中釋放出來。其中，修飾 DNA 之磷酸葡萄糖胸苷轉移酶的活性最佳，較直接固化以及透過 DNA 固化在磁性奈米粒子的活性好；另外，無論是以直接固化或是透過 DNA 固化的方式，半乳糖激酶的活性都相似。透過加熱從奈米粒子釋放出具有 DNA 之酵素，可利用磁性奈米粒子再回收利用。

關鍵字

蛋白質修飾 ； 蛋白質固化 ； 去氧核醣核酸 ； 氟化物 ； 磁性奈米粒子

並列摘要

Chemical modification of protein is an important tool for studying protein structure and function. To avoid the loss of protein activity, site-specific protein modification has been extensively studied in last decades. The studies of this thesis focused on the developments of site-specific protein modification and immobilization of modified protein on solid support by fluorous- or DNA-tagged protein.
The unique affinity interaction between fluorous molecules has been applied in many fields. We took advantage of the resistance of non-specific interaction by fluorous surface on protein microarray fabrication. Two strategies for site-specific modification of proteins with a fluorous tag were developed in this thesis. First, the target protein, enhance green fluorescent protein (eGFP), maltose binding protein (MBP), and glutathione transferase (GST), were expressed by intein expression system and their C-terminus were conjugated with cysteic acid contained fluorous tag by native chemical ligation (NCL). Second, the anti-RAC antibody was labeled with boronic acid contained fluorous tag through boronic acid-diol interaction. The fluorinated protein were site-specifically immobilized on fluorous solid support by simply mixing the fluorinated protein and solid support. The non-covalent fluorous-fluorous interaction were stable enough to withstand continuous washing and presented excellent performance to suppress the non-specfic adsorption.
DNA biosensor technologies are currently under intense investigation owing to their great promise for rapid and low-cost detection of specific DNA sequence. In this thesis, the specific interaction between DNA base pairs was applied on the protein immobilization on the magnetic nanoparticles to investigation of the activity difference between free and immobilized enzymes. The target enzyme, RmlA and GalK, were site specifically modified with DNA at their C-terminus using 2-cyanobenzothiazole (CBT)-cysteine (Cys) condensation reaction to give Enzyme-DNA. Then, these enzymes were assembled on DNA@MNPs through the sequence-specific hybridization properties of DNA. The captured enzymes were released from DNA@MNPs when the incubation temperature was higher than Tm of dsDNA. The results showed that the activity of RmlA-DNA is higher than those of RmlA-DNA-DNA@MNP and directly immobilized RmlA@MNP. However, the activity of GalK is identical as those of GalK-DNA-DNA@MNP and directly immobilized GalK@MNP. The enzyme-DNA was easily recovered by incubation with DNA@MNP and can be re-used after released from MNP by heating.

並列關鍵字

protein modification ； protein immobilization ； DNA ； fluorous ； magnetic nanoparticle

參考文獻

128.蕭偉鎮 結合化學與酵素方法合成多醣體. 民國 103年, 國立清 華大學化學研究所 博士論文.

125.侯凱齡 利用氟標記輔助化學及酵素合成寡醣. 民國102年, 國立 清華大學化學研究所 碩士論文.

129.簡薇庭 階段型合成醣核苷與其應用於合成聚 N-乙醯乳醣胺. 民 國 101 年, 國立清華大學化學研究所 博士論文.

126.李泗芃 探討噬熱菌 Meiothermus taiwanensis ATCC BAA-400 之半乳糖激酶酵素動力學及應用於合成Pk抗原類似物. 民國101 年, 國立清華大學化學研究所 碩士論文.

127.郭力禎 酵素唾液酸化 Globo- 系列醣體. 民國104年, 國立清華 大學化學研究所 碩士論文.