- 學位論文
聚球藻RF-1之97-kDa鈣結合蛋白的研究
Studies on the 97-kDa calcium-binding protein in Synechococcus RF-1
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摘要
聚球藻RF-1細胞中具有一分子量為97-kDa的蛋白質，以45Ca2+標識法鑑定以後發現，其為該藻最主要之鈣結合蛋白。經以SDS萃取液或Tween-20萃取液抽取，發現其含量相當恆定，但培養基中缺鈣會造成其含量降低。經缺鈣 (加EGTA) 處理後，可在native PAGE觀察到氧化及還原兩種型態，而含鈣處理 (不加EGTA) 則可見到多個蛋白質帶，推測它可能具有多個鈣結合位。為鑑定合成此蛋白的基因，利用其N端胺基酸序列合成的退化性寡核甘酸 (degenerated oligonucleotides) 作為探針，與限制酵素切割過的聚球藻RF-1的DNA 進行南方轉漬分析，可偵測到一段2.2 kb EcoRI-HindIII DNA片段含有雜合訊號，由DNA序列分析之結果證明此段DNA含部份N端開始之鈣結合蛋白基因。以PCR技術合成含鈣結合蛋白部份基因的DNA探針，並以pUC19或λZAPⅡ為載體，無法偵測到含完整鈣結合蛋白基因的選殖體；然而以pUC19為載體選殖HindIII切割聚球藻RF-1 DNA之3 kb片段，則篩選到一個含6 kb插入DNA的選殖體，此段插入DNA包含部份鈣結合蛋白基因的DNA序列。以pUC19及pBR322重新選殖此選殖體的插入DNA，發現6 kb之選殖體是由二段3 kb片段接合後插入pUC19中，其中一段為部分鈣結合蛋白基因，另一段不是。實驗亦證明含部份鈣結合蛋白基因之3 kb DNA片段似乎無法單獨選殖於pUC19，而需一段其他的DNA片段作為輔助。此段3 kb DNA片段所含鈣結合蛋白基因之DNA序列較先前2.2 kb DNA片段長，若以此延伸之部份DNA作為探針，應可選殖到含此蛋白質C端部份之DNA序列，並進而得到完整鈣結合蛋白基因之DNA序列。
並列摘要
In cyanobacterium Synechococcus RF-1 a 97-kDa protein was identified by 45Ca2+ autoradiography being the most predominant calcium-binding protein. This protein was extractable in both the SDS sample buffer and the Tween-20 buffer. Under the implemented culture condition, the abundance of the 97-kDa protein in Synechococcus RF-1 remained constant but was reduced upon depletion of calcium ion in the medium. With the presence of EGTA, the 97-kDa calcium-binding protein migrated into a reduced isoform and an oxidized isoform in a native gel. With the absence of EGTA, the 97-kDa calcium-binding protein was separated into several distinct bands in the native gel, suggesting that it might reside multiple calcium binding sites. To identify the gene encoding the 97-kDa protein, the Synechococcus RF-1 genomic DNA was restriction enzyme-digested and analyzed by Southern blotting using a degenerated oligonucleotide probe corresponding to the N –terminus amino acid sequence of the 97-kDa protein. A 2.2 kb EcoRI-HindIII cut DNA fragment has been cloned and sequenced. It was identified to contain the DNA sequence of the first 108 amino acids from the N-terminus of the protein. Attempts to clone a 6 kb Xba I cut fragment of I cut DNA fragments into the expression vector of pUC19 and lZAPII have not been successful and this is plausibly incurred by the lethal effect of the expressed calcium binding protein to the host cells. So another strategy was used. Based on the restriction map, the DNA sequence of the complete gene can be identified by cloning two overlap DNA fragments. A 3 kb HindIII-cut DNA fragment was cloned into pUC19, and one clone containing the target DNA has been identified. However, besides the target DNA fragment, the clone seemed to contain a different insert DNA of 3 kb. The suggestion was proved when the insert DNA was subcloned into a non-expression vector pBR322. When the target DNA fragment is sequenced, it will be used as a probe to clone the C-terminal region of the calcium-binding protein gene. Further sequencing experiment would help unravel the complete genetic sequence of the 97-kDa calcium binding protein.
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