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研究生:陳靜瑩
研究生(外文):Chen Ching-Ying
論文名稱:牛去氧核醣核酸水解之cDNA選殖及兩個結構性鈣離子結合位置之功能分析
論文名稱(外文):Bovine pancreatic deoxyribonuclease:cDNA cloning and the functional roles of the two structural calcium sites
指導教授:廖大修
指導教授(外文):Liao Ta-Hsiu
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:生化學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:英文
論文頁數:117
中文關鍵詞:去氧核醣核酸水解鈣離子結合位置必要雙硫鍵胰蛋白去活性定點突變雙股水解
外文關鍵詞:deoxyribonucleasecalcium-binding sitesessential disulfidetrypsin inactivationsite-directed mutagenesisdouble scission
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牛胰臟去氧核糖核酸水解(bpDNase)之cDNA經選殖後加以定序。bpDNase的cDNA含有1295個核酸,可轉譯出一個有282個胺基酸的多鏈,此多鏈除包含成熟的bpDNase胺基酸序列外,其N端還多了一段由22個胺基酸所組成的疏水性訊息。進一步我們合成了蛋白質表現專用的cDNA,此cDNA序列除了可以表現成熟的bpDNase 260個胺基酸外,在其N端還多了Met與Ala兩個胺基酸。將此段cDNA送入表現載體pET15b後,先以E. coli DH5a作為宿主,篩選含正確cDNA表現序列的殖株,再抽取殖株的質體,送入E. coli BL21(DE3)pLysE,用IPTG誘導brDNase表現後,可在培養液中測出DNase活性。利用Mono Q管柱與鈣離子梯度濃度析出法進行純化。純化的brDNase其分子量約為29 kDa。當比較brDNase與bpDNase水解DNA的速率時,得到有相似的比活性。經胺基酸序列分析後,得知brDNase第一個胺基酸為Ala。而因Asn19可被測得,故可確定此重組蛋白質無醣化修飾。
利用定點突變法,繼續研究brDNase的數個重要胺基酸的重要性。首先將brDNase催化中心的His134改為Gln。此brDNase(H134Q)仍可利用鈣離子梯度濃度析出法進行純化,而且所悉出之位置與brDNase沒有差異,可見brDNase(H134Q)仍保有兩個鈣離子結合位置,然而此蛋白質已失去水解DNA的活性。
另外我們也分別對兩個結構性鈣離子結合位置進行定點突變。及把Asp99及Asp201改變為Ala。經蛋白質表現及純化,先對突變蛋白質進行鈣離子結合強度分析。結果得知,與bpDNase比較,突變蛋白質brDNase(D99A)與brDNase(D201A)分別有一個鈣離子結合位置被破壞。以大分子DNA為水解受質,比較水解活性後發現brDNase(D99A)仍保有bpDNase的61 %水解活性,其Vmax值不改變只有Km值上升。而brDNase(D201A)僅保有13 %水解活性,此時其Km值上升且Vmax值下降。以小分子NPPP為水解受質比較水解活性,則brDNase(D99A)保有bpDNase的33 %水解活性,而brDNase(D201A)保有25 %水解活性。以限制重組置換DNA片段,建構出擁有雙突變(D99A/D201A)的表現質體pETDM,利用西方墨點法及活性染色法,測定brDNase(D99A/D201A)的比活性,發現雙突變蛋白質仍保有bpDNase水解活性的1.2 %。
當鎂離子與鈣離子同時存在或是錳離子單獨存在,對bpDNase與brDNase(D99A)而言,可擁有切斷DNA雙股的能力,brDNase(D201A)則喪失此能力。在10 mM鈣離子存在時,brDNase(D99A)與bpDNase均可被鈣離子保護,不被胰蛋白水解,只有brDNase(D201A)會被胰蛋白水解。但以β-MSH處理時,在10 mM鈣離子存在下,bpDNase及兩種突變蛋白質都可被鈣離子保護,不被β-MSH還原而喪失活性。利用螢光光譜分析與紫外光差異光譜測定分析可得知,當bpDNase及兩種突變蛋白質結合上鈣離子時,都會造成蛋白質的構形改變。綜合以上結果可推論當鈣離子與兩個結構性鈣離子結合位置結合,跟蛋白質構形改變及保護酵素必要雙硫鍵不被β-MSH打開的機制無關,但與保護DNase I不被胰蛋白水解及酵素活性的調節有關。

The bovine pancreatic (bp) DNase gene was cloned from bp cDNA and expressed in E. coil. A polynucleotide sequence of 1295-base was deduced from clones of the cDNA. The sequence showed an open reading frame, which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bpDNase. An expression plasmid was constructed by inserting into the vector pET15b a cDNA fragment coding for bpDNase ligated with a hexanucleotide coding for Met-Ala at the 5'-end. The plasmid was transformed into E. coli strain BL21(DE3)pLysE and the active bovine recombinant DNase (brDNase) was produced after induction of protein synthesis. From the induced culture medium, brDNase was purified with a Mono Q column. The purified brDNase shows a molecular mass of 29 kDa and has the same specific activity as does bpDNase. The NH2-terminus of brDNase is Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bpDNase, Asn18 is non-glycosylated.
Using site-directed mutagenesis, we changed the brDNase active site His134 to Gln. The brDNase(H134Q), like bpDNase, was purified on a Mono Q column based on the principle of calcium affinity elution, indicating the retention of the two Ca2+-binding sites. The mutant protein thus obtained was not active.
The two amino acid residues, Asp99 and Asp201, which involved in the coordination of the two calcium atoms found in the X-ray structure of bpDNase, were individually changed by site-directed mutagenesis. The two altered proteins, brDNase(D99A) and brDNase(D201A), expressed in E. coli, were purified by Ca2+-affinity chromatography. Equilibrium dialysis showed that mutation destroyed one Ca2+-binding site each in brDNase(D99A) and brDNase(D201A). Compared to bpDNase, when the large molecular DNA was used as substrate, the Vmax value for brDNase(D99A) remained unchanged and that for brDNase(D201A) was decreased, while the Km values for the two variants were increased 2-3 fold. When the small molecular NPPP was used as substrate, the brDNase(D99A) had 33 % of the bpDNase activity remaining and the brDNase(D201A), 25 %. Restriction enzymes were used to cut pETD99A and pETD201A and the products were used to construct a double mutant (D99A/D201A) plasmid, pETDM. This plasmid expressed a brDNase (D99A/D201A) which had, based on Western blotting and activity staining, 1.2 % of the bpDNase activity remaining.
Like bpDNase, brDNase(D99A) was able to make double-scission on duplex DNA with Mg2+ plus Ca2+ and was effectively protected by Ca2+ from the trypsin inactivation. But under the same conditions, brDNase(D201A) lost the double-scission ability and was not protected by Ca2+. Nevertheless, the two variant proteins retained the characteristics of the Ca2+-induced conformational changes and the Ca2+-protection against the β-mercaptoethanol disruption of the essential disulfide bond, suggesting that other weaker Ca2+-binding sites not found in the X-ray structure were responsible for these properties. Therefore, the two structural calcium atoms are not for maintaining the overall conformation of the active DNase, but rather play the role in the fine-tuning of the DNase activity.

中文摘要------------------------1
英文摘要------------------------ 4
縮寫------------------------------7
緒論------------------------------9
實驗材料與儀器--------------- 13
實驗方法------------------------16
結果------------------------------50
討論----------- -------------------62
圖表與圖表說明---------------73
參考文獻------------------------ 112

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