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首页> 外文期刊>Journal of bacteriology >Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii.
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Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii.

机译：梭状芽胞杆菌NADP连接的7α-羟基类固醇脱氢酶基因的表征和调控。

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A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group.
机译：通过离子交换，凝胶过滤和染料-配体亲和层析将得自梭状芽胞杆菌ATCC 9714的胆汁酸诱导的NADP连接的7α-羟基类固醇脱氢酶（7α-HSDH）纯化310倍。纯化的酶的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示一条主要的肽带（30,000 Da）。确定了N-末端序列，并合成了相应的寡核苷酸，并用于筛选C. sordellii的EcoRI和HindIII基因组消化物。随后通过与pUC19连接并转化到大肠杆菌DH5α-MCR中来克隆两个分开的片段（4,500 bp，EcoRI; 3,200 bp，HindIII）。显示了EcoRI片段包含一个截短的7α-HSDH基因，而HindIII片段包含了整个编码区。含有HindIII插入片段的大肠杆菌克隆表达了高水平的NADP连接的7α-HSDH。核苷酸序列分析表明，7α-HSDH由单顺反子转录单元编码，其DNA序列元件类似于位于上游和下游侧翼区域的不依赖于rho的终止子。通过引物延伸分析定位转录起始位点。 Northern（RNA）印迹分析表明，响应于生长培养基中胆汁酸的存在，诱导在转录水平上介导。另外，在未诱导的培养物中观察到生长期依赖性表达。对预测的蛋白质序列的分析表明，该酶可以分类为短链脱氢酶组。

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