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標題: Bacillus sp. P-6中性金屬蛋白基因的選殖、定序及其基因特性之探討
Cloning, Sequencing and Characterization of Neutral Metalloprotease Gene from Bacillus sp. P-6
作者: 陳韻帆
Chen, Yun-Fan
關鍵字: Cloning;基因的選殖定序;Sequencing;Characterization;Neutral Metalloprotease Gene;Bacillus sp.;pro-sequnece function;基因特性;中性金屬蛋白;枯草桿菌屬
出版社: 食品科學系
本研究藉由shotgun-cloning方式,以E. coli DH5α為宿主細胞pBluescript II KS(-)為載體構築一Bacillus sp. P-6基因庫,並由一萬多株轉型株中篩選得到一具有蛋白活性之選殖株,此選殖株質體命名為pBBNP-7。由DNA序列分析結果顯示,此選殖基因片段含有一1,698 bp 的open reading frame (ORF) 可轉譯出566個胺基酸,推算其分子量約為63 kDa。此轉譯出的蛋白質經胺基酸序列比對結果預測可分為三部份,分別為N端的pre-sequence(signal peptide)、pro-sequence 和mature protease,其中預測的mature protease 與野生株所純化出的protease分子量相同,西方墨漬法結果亦相符。將選殖出的DNA片段,依所包含之上述三個多部份,分別構築成重組表現載體pET2PM, pETProM, pETM,於E. coli中以低溫誘導蛋白質,實驗中只有E. coli BL21(DE3)/pET2PM之上清液具有蛋白活性(40 U/ml),而E. coli BL21(DE3)/pETProM、E. coli BL21(DE3)/pETM之上清液則不具蛋白活性,此結果顯示pre-sequence與pro-sequence兩者對於活體內(in vivo)蛋白之熟成(processing)缺一不可,且蛋白之熟成作用應在蛋白質送至胞壁空間後才進行。另構築雙向載體pHBNP,將完整蛋白基因包含原有的啟動子送入Bacillus subtilis DB430進行表現,於LB培養基中培養24小時後,上清液中之蛋白活性約95 U/ml;在營養限制之SSY培養基中培養36小時則可得115 U/ml之蛋白活性。

Employing pBluescript II KS(-) as a cloning vector, the genomic library of Bacillus sp. P-6 was constructed in E. coli DH5a by way of shotgun-cloning approach. A candidate possessing protease activity was screened among approximately ten thousands of colonies by skim milk contained plate. The recombinant plasmid of the candidate was named as pBBNP-7. Analyzing the DNA sequence of the inserted DNA fragment, a 1,698 bp open read frame encoded a 566 amino acid of protein was predicted and its putative molecular mass was 63 kDa. According to the amino acid sequence alignment, the primary structure of deduced protein could be classified into three portions, since from N terminus, pre-sequence, pro-sequence, and mature protease. The deduced molecular mass of mature protease was consistent with the protease purified from Bacillus sp. P-6. and also validated by Western blotting. In the protein expression experiment at low temperature, protease activity was detected (40 U/ml) in the culture broth in E. coli BL21(DE3)/pET2PM, however, there were no protease activity detected in E. coli BL21(DE3)/pETProM and E. coli BL21(DE3)/pETM. These results suggested that pre-sequence and pro-sequence were simultaneously required for protein maturation in vivo, and the maturation was occurred after protein secreted into periplasmic space. A protease expression vector pHBNP, bearing native promoter and capable of shuttling E. coli and Bacillus subtilis, was also constructed in this study. Cultivated the Bacillus subtilis DB430/ pHBNP in LB medium, there was a maximal 95 U/ml of protease activity detected. While, a maximal 115 U/ml of protease activity detected, cultivated in nutrient limited medium SSY.
Appears in Collections:食品暨應用生物科技學系

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