發展多效桿狀病毒表現載體並用以生產具功能性Neuroligin 1的胞外片段及NR1A/NR2B△C NMDA受體蛋白之研究

Development of polycistronic baculovirus expression system and employ it to produce functional neuroligin-1 extracellular fragment and NR1/NR2B△C receptors in insect cells

陳文碩(Wen-Shuo Chen)

指導教授 ： 張兗君 ； 吳宗遠

國立清華大學/生命科學院/分子醫學研究所/博士(2011年)

摘要

神經細胞黏附蛋白，neuroligin 及neurexin的交互作用為連接前突觸與後突觸神經細胞所必需，並具有調控神經訊號傳遞及特化神經之功能性。Neuroligins (NLs) 被發現在後突觸細胞膜上具有促進神經軸突生成的能力。在第參章中，我們使用雙效桿狀病毒表現系統來同時表現NL1的胞外片段蛋白與綠螢光蛋白。重組的NL1蛋白會分泌至細胞上清液中，而且利用胞內綠螢光釋放至胞外做為細胞裂解的訊號，用來決定最佳收取重組NL1蛋白的時間點。我們從感染的細胞上清液中，利用鎳管柱進行親和層析純化後，可得到 96 %純度以上的重組NL1蛋白，產量約129 ± 13 μg / 8x107 High Five cells。我們藉由轉印在玻璃基板上處理NL1/PLL (poly-L-lysine) (137.07 ± 9.74 μm/day) 以及PLL (105.53 ± 4.53 μm/day) 的實驗來證實純化後的NL1蛋白的確具有促進神經軸突的生長速率之活性。這些結果也經由免疫螢光染色來證實及指出重組蛋白可經由一步驟親和性層析與利用雙效桿狀病毒表現系統來監測細胞裂解純化而得。此技術及此玻璃基板方法學可提供一個簡單、有效及有用的平台來了解在神經再生與突觸形成的研究上，外來因子所扮演的角色。
在大腦中，NMDA受體不僅是興奮性神經傳導的重要調節者，而且在長期增益作用 (long term potentiation) 上也扮演重要的角色。NMDA受體是一種對鈣離子具高通透性的離子通道，過度活化會導致神經細胞的死亡。先前研究指出具有甘胺酸結合位子的NR1次單元蛋白需與 NR2或NR3次單元蛋白共同組裝 (assembly) 才能形成具有功能性的NMDA受體。然而，NMDA受體的四級結構仍尚未被建立。為了要表現具有功能性的NMDA受體，多效真核表現系統是必須的。在第肆章中，我們藉由IRESs的連接構築出三效桿狀病毒表現載體，並證實可同時表現三個基因。在伍章中，我們利用三效桿狀病毒表現載體於昆蟲細胞中同時表現NMDA受體蛋白。使用西方轉漬法及免疫螢光染色來證實重組之NMDA受體可在膜上表現。鈣離子通道活性分析的結果指出受感染的昆蟲細胞膜上的NMDA受體會受到麩胺酸及NMDA的刺激而導致胞內鈣離子濃度的變化。這些研究對NMDA受體的蛋白結構上的決定是有所助益的。

關鍵字

桿狀病毒 ； 核醣體內轉譯序列 ； 三效載體 ； Neuroligin-1 ； NMDA受體

並列摘要

The interaction between the synaptic adhesion molecules neuroligins and neurexins is essential for connecting the pre- and post-synaptic neurons, modulating neuronal signal transmission, and facilitating neuronal axogenesis. In chapter 3, we describe the simultaneous expression of the extracellular domain of neuroligin 1 (NL1) proteins along with the enhanced green fluorescent protein (EGFP) using the bi-cistronic baculovirus expression vector system (bi-BEVS). Recombinant NL1 proteins were secreted into the culture medium and the optimum harvest time for NL1 proteins based on the lysis of infected cells was determined by the release of cytosolic EGFP. The NL1 protein (129 ± 13 μg / 8x107 High Five cells; ~96% purity by metal affinity chromatography) was obtained from the supernatant of the recombinant virus-infected insect cells. A novel chip was employed to address whether the recombinant NL1 is functional in axogenesis. The purified NL1 promoted and enhanced the growth rate (137.07 ± 9.74 μm/day) of the axon on NL1/PLL (poly-L-lysine)-coated fine lines on the chip compared to those lines that were coated with PLL alone (105.53 ± 4.53 μm/day). These results were confirmed by fluorescence immunocytochemistry and demonstrated that the recombinant protein can be purified by a one-step process using IMAC combined with monitoring of cell lysis by bi-BEVS. This technique along with our novel chip offers a simple, cost-effective and useful platform for understanding the roles of NL1 protein in neuronal regeneration and synaptic formation studies.
NMDA receptors are critical mediators of excitatory neurotransmission in the brain, being pivotal for long term potentiation. NMDA receptor channels are highly permeable to calcium ions, and thus overactivation leads to excitotoxic neuronal cell death. Functional NMDA receptors are formed from the co-assembly of the obligatory NR1 glycine-binding subunit with NR2 and/or NR3 subunits. However, the quaternary structure of NMDA receptors is still not yet established. To functional express these important heteromeric NMDA receptors, a polycistronic eucaryotic expression system is required. In chapter 4, we had constructed a tri-cistronic expression votor by the inclusion of two internal entry sites (IRESs) and demonstrated that three genes can be co-expressed by this technique. In chapter 5, we employed the novel baculovirus expression vectors to express NMDA receptors in Sf21 insect cells. Western blot and immunostaining demonstrated the expression of NMDA receptors on plasma membrane. Calcium image analysis indicated the function of this recombinant NMDA is responsible to NMDA and glutamate. These studies will facilitate the protein structure determination of NMDA receptor.

並列關鍵字

Baculovirus ； IRES ； Tri-cistronic vector ； Neuroligin-1 ； NMDA receptors

參考文獻

Al Ahmad, A., Lee, B., Stack, J., Parham, C., Campbell, J., Clarke, D., Fertala, A. and Bix, G.J., Endostatin binds nerve growth factor and thereby inhibits neurite outgrowth and neuronal migration in-vitro. Brain Res 1360, 28-39.

Ali, S.A., Joao, H.C., Csonga, R., Hammerschmid, F. and Steinkasserer, A., 1996. High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization. Biochem J 319 ( Pt 1), 191-5.

Anegawa, N.J., Guttmann, R.P., Grant, E.R., Anand, R., Lindstrom, J. and Lynch, D.R., 2000. N-Methyl-D-aspartate receptor mediated toxicity in nonneuronal cell lines: characterization using fluorescent measures of cell viability and reactive oxygen species production. Brain Res Mol Brain Res 77, 163-75.

Arac, D., Boucard, A.A., Ozkan, E., Strop, P., Newell, E., Sudhof, T.C. and Brunger, A.T., 2007. Structures of neuroligin-1 and the neuroligin-1/neurexin-1 beta complex reveal specific protein-protein and protein-Ca2+ interactions. Neuron 56, 992-1003.

Awobuluyi, M., Yang, J., Ye, Y., Chatterton, J.E., Godzik, A., Lipton, S.A. and Zhang, D., 2007. Subunit-specific roles of glycine-binding domains in activation of NR1/NR3 N-methyl-D-aspartate receptors. Mol Pharmacol 71, 112-22.