重組綠竹 Monocot-Specific Protein-41 之純化與性質檢定

Purification and characterization of the recombinant Bambusa oldhamii Monocot-Specific Protein-41

李佩儒(Pei-Ju Lee)

指導教授 ： 王愛玉

共同指導教授 ： 楊健志(Chien-Chih Yang)

國立臺灣大學/生命科學院/生化科技學系/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10553

摘要

BoMSP41 (Monocot-specific protein-41 in Bambusa oldhamii) 為綠竹之未知功能基因，其基因表現在綠竹生長快速時期之節間部分會大幅提升。此基因轉譯出的蛋白質BoMSP41被預測為一個內生性不具穩定構型的蛋白質 (intrinsically disordered protein, IDP) 以及可能會被運送至成熟的葉綠體中。為了瞭解BoMSP41的特性，本研究用大腸桿菌中表現帶有 His-tag 的重組BoMSP41蛋白質並進行純化。重組BoMSP41被觀察到具有一些特殊的性質，包括：在SDS 膠體上的泳動率比預期的分子量應有的泳動率低、以膠體過濾層析預測的分子量高於預期的分子量以及具熱穩定性等，這些特性也是在IDPs中常被觀察到的特性。將純化過的重組BoMSP41利用圓二色光譜 circular dichroism (CD) 觀察其二級結構，結果顯示BoMSP41的二級結構中有8.6% alpha-helix、 37.0% beta-sheet、12.9% beta-turn 及 41.5% random coil。由上述結果顯示BoMSP41為具有disordered region，符合之前所預測的結果。以純化過後的重組BoMSP41蛋白質為抗原製備多株抗體，並對快速生長中的綠竹筍蛋白質粗抽液進行西方墨點法分析，結果顯示BoMSP41基因會在植物體內轉譯出BoMSP41蛋白質，而且可能不存在於葉綠體或細胞核分劃中。BoMSP41可能需要與其它蛋白質結合，以使其不具構形的區域轉變為具穩定的結構，以執行其功能。

關鍵字

綠竹 ； 單子葉植物特有蛋白質 ； 重組蛋白質 ； 包涵體 ； 純化 ； 圓二色光譜 ； 內生性不具穩定構型蛋白質 ； 細胞內定位

並列摘要

BoMSP41 (Monocot-specific protein-41 in Bambusa oldhamii), an unknown function gene, is highly up-regulated in the internode-containing region of rapidly elongating bamboo shoots. The protein encoded by this gene was suggested to be an intrinsically disordered protein (IDP) and might be translocated into chloroplasts. To understand the characteristics of BoMSP41, the His-tagged recombinant BoMSP41 proteins produced in E. coli were purified. The recombinant BoMSP41 had several unusual properties including a lower electrophoretic mobility on SDS-PAGE, overestimated molecular weight with gel filtration and high thermal stability, which are generally observed in many IDPs. Analysis of the purified recombinant BoMSP41 by circular dichroism (CD) spectroscopy suggested that there were 8.6% alpha-helix, 37.0% beta-sheet, 12.9% beta-turn and 41.5% random coil in BoMSP41. The results showed that BoMSP4 contained intrinsically disordered regions as previously predicted. The purified recombinant BoMSP41 proteins were used as antigen to raise polyclonal antibodies. Western analyses of proteins extracted from developing bamboo shoots with the polyclonal antibodies showed that the transcripts of BoMSP41 gene could be translated into proteins in planta and BoMSP41 might not be present in chloroplasts and nuclei. To perform its function, BoMSP41 might require binding partners to induce the transition of its disorder region to ordered conformation.

並列關鍵字

bamboo ； monocot-specific protein ； recombinant protein ； inclusion bodies ； purification ； circular dichroism ； intrinsically disordered protein ； subcellular localization

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