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Title: 綠豆澱粉磷解脢的基因選殖、表現與生化特性探討
Cloning, Expression and Molecular Kinetics of Mungbean (Vigna radiata L.) Starch Phosphorylase
Authors: 柯源悌;賴建成
Contributors: NTOU:Department of Food Science
Keywords: 澱粉磷解?;重組蛋白;蛋白質複合物
starch phosphorylase;recombinant protein;protein complex
Date: 2010-08
Issue Date: 2011-06-28T05:52:18Z
Publisher: 行政院國家科學委員會
Abstract: 摘要:提出三年計畫對先前所鑑定出的綠豆105-kDa澱粉磷解酶(starch phosphorylase, SP, EC、曾經偵測其轉譯後修飾、會與SP結合的複合蛋白的初步證據,提出一個在 綠豆澱粉生合成路徑可能由一個Glc-1-P pathway所主導的假說,作進一步的分子探討。 第一~二年將選殖出綠豆SP異構型的cDNA全長,在宿主內表現重組蛋白,進行特性分 析。先設計基因專一性引子用RT-PCR、RACE (rapid amplification of cDNA ends)方法選 殖出cDNA的全長,將全長或截短、雜合方式分別放入原核或真核的表現載體系統中表 現重組SP蛋白,分析表現含量與活性的適量化。接著,進行大量製備與純化重組SP,製 備抗體作免疫分析使用,同時進行生化特性如適當pH、適當溫度、安定性等的基礎研究, 評估基質偏好性、調節因子作分別的探討。第二年也將著重建立其直鏈葡聚多醣產物的 分析方法,評估SP成為功能性酵素的潛力。第三年研究SP結合複合蛋白的分子動力學, 追加SP的角色。將利用重組SP製作抗體,使用免疫沉澱,在不同的生長階段、組織部位、 細胞區分或胞器等,抓出與SP結合的蛋白質複合物,利用質譜儀鑑定出複合物的蛋白組 成;再製備複合物,評估在試管內合成多醣的能力,藉由產物與中間代謝物的分析,與 現階段澱粉合成與代謝流程,推演出綠豆SP 的角色,驗證Glc-1-P pathway 假說。同時 探討是否SP 在被修飾之下,調節澱粉的生合成。利用蛋白質體分析技術,以二維電泳 和質譜儀,分析SP分子上具有被修飾的官能基,辨別出是否修飾或不同修飾程度的SP 狀態,會影響活性、構形與被調節的分子機制存在。
Abstract:A three-year project is proposed here for the molecular investigations of a hypothesized Glc-1-P pathway in mungbean starch biosynthesis based on previous findings of mungbean starch phosphorylase (SP, EC which have identified, and preliminarily detected its post-translational modification and co-existing protein complexes. The first two years plan to clone and express recombinant SP for molecular characterization. Gene-specific primers will be designed for RT-PCR and RACE (rapid amplification of cDNA ends) to clone the full length cDNA of SP isoforms and to be expressed in full, truncated or chimeric forms in proper prokaryotic or eukaryotic hosts. Biochemical properties such as enzyme activity, pH and temperature stability, substrate preference, regulatory cofactors and the optimal production parameters will be performed on recombinant SP. The second year will also establish analytical and quantitative methods for the linear glucan polymeric SP products, metabolic intermediates and evaluate its potential use as a functional enzyme. The third year will study the molecular kinetics of the protein complexes SP associated under normal physiological condition at different mungbean growth stages, tissues, cellular localizations. It will reveal the roles of SP may play in starch biosynthesis and the hypothesized Glc-1-P pathway. The recombinant SP will be prepared to generate SP specific antibodies. Immunoprecipitation using SP-specific antibodies will be conducted to capture the complexes to identify the protein components by Mass spectrometry. The protein complex will be prepared to test its ability to synthesize glucan polymers in vitro. In addition, proteomic analysis by 2-dimensional electrophoresis and Mass will be used together to study where the molecular location, types and degree of post translational modifications on SP in detail. Prospective results would clarify whether such modification required to modulate SP activities and conformation or certain component of the complexes is under a cellular controlled mechanism in the molecular level.
Relation: NSC97-2313-B019-011-MY3
Appears in Collections:[Department of Food Science] Research Reports

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