蛋白質(zhì)元器件的合成與組裝

冷艷　門冬

摘 要：利用合成生物學(xué)建立人工合成生物體系與生物制造是一個(gè)系統(tǒng)工程，人造功能器件在其中發(fā)揮著重要作用。當(dāng)前的合成生物學(xué)能夠組織不同來源的順序催化酶分子在細(xì)胞內(nèi)制造全新的合成途徑，但仍缺少在空間上有機(jī)組織酶分子的手段。如果能夠人為地構(gòu)建空間有序的多酶復(fù)合體將使人工合成反應(yīng)體系更快速、有效。蛋白納米纖維和病毒顆粒及其包裝蛋白區(qū)別于其它納米材料，具有天然的蛋白質(zhì)特性、穩(wěn)定規(guī)整的結(jié)構(gòu)和自組裝特性，可用構(gòu)建人工蛋白“腳手架”來“鉚定”多酶反應(yīng)體的功能元件，以保護(hù)多酶耦合體系并實(shí)現(xiàn)其順序裝配。該項(xiàng)目一方面通過基因克隆構(gòu)建了帶有功能元件的自組裝元件，發(fā)展了一種自組裝功能納米纖維的制備方法，并利用不同熒光信號(hào)分子標(biāo)記代表不同蛋白組分，實(shí)現(xiàn)多種不同標(biāo)記的Sup35有序組裝。同時(shí)，發(fā)展了一種基于磁珠技術(shù)控制的催化納米線的制備方法，該方法不同于傳統(tǒng)的吸附、包埋和共價(jià)交聯(lián)等方法，其偶聯(lián)過程依賴于自組裝，不會(huì)產(chǎn)生形變、變性或取向性明顯改變，因此對(duì)酶分子本身的活性少有直接的影響。對(duì)酶分子的固定化和多酶催化體系的模擬提供了新的技術(shù)路線和思路。另一方面以SV40病毒衣殼蛋白和HBV衣殼蛋白為研究對(duì)象，獲得了兩種體外組裝的類病毒樣顆粒，這兩種類病毒樣顆粒都具有籠型蛋白結(jié)構(gòu)。這種籠型蛋白結(jié)構(gòu)是空心的核殼性結(jié)構(gòu)，一般具有內(nèi)腔，這些特點(diǎn)為制造多層級(jí)、復(fù)雜、順序的多酶體系提供了優(yōu)良的性質(zhì)。該研究利用這個(gè)特性，在不影響類病毒樣顆粒組裝結(jié)構(gòu)的前題下，在結(jié)構(gòu)表面插入了一些功能基團(tuán)，實(shí)現(xiàn)了這種三維蛋白骨架的多功能化。

關(guān)鍵詞：人造功能器件 蛋白納米纖維 類病毒樣顆粒 組裝 多功能化

Abstract：Using of synthetic biology to establish artificial synthetic biological systems and bio manufacturing is a systematic engineering，in which artificial function device plays a very important role. With Present synthetic biology technique scientists can to make use of sequential catalytic enzymes from different sources to build new synthetic pathways in cells， but scientists still lack the means to narrow the distance between the enzyme molecules to each other in space. Synthetic reaction system will become more rapid and more effective if we can artificially build space orderly multienzyme system. Different from other nano materials， protein nanofiber and virus-like particle， which have characteristics of natural protein， regular and stable structure and self-assembly properties， can be used to construct artificial protein “scaffolding” for “riveting” multienzyme system. This artificial protein "scaffolding" protect enzyme coupling system and its sequence assembly. In this project， on one hand， we construct the self-assembling component with functional components by gene cloning， develop the method for preparation of self-assembling functional nanofiber， and make use of different fluorescence signal molecular markers to represent different protein components for achieving ordered assembly of different labeled sup35 protein. At same time， we develop the method for preparation of the catlytic nanowires based on control of magnetic bead technique.Different from the traditional method of adsorption， entrapment and covalent cross-linking， the coupling process in this method relies on self-assembly， and deformation， degeneration or orientation change will not generate， so that only little direct influence to the activity of enzyme molecule will appear. This method provides a new technology roadmap and ideas for immobilization of enzyme molecules and simulation of multienzyme system. On the other hand， through the study of SV40 capsid protein and HBV capsid protein， we obtain two self-assembly of virus-like particles in vitro. Virus-like particles have cage-type protein structure， which is hollow core-shell structure and has cavity generally. These characteristics of virus-like particles provide excellent properties for manufacturing multi-level， complex， sequential multienzyme system and achieving multi-functional three-dimensional protein.

Key Words：Artificial function device；Protein nanofiber；Virus-like particles；Assembly；Multi-functional

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