A study on a mechanism for the transcriptional regulation of an epsilon-toxin gene by a novel type of bent DNA

新型弯曲DNA对ε-毒素基因转录调控机制的研究

基本信息

批准号：
18590428
负责人：
OKABE Akinobu
金额：
$ 2.47万
依托单位：
Kagawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

An epsilon-toxin gene of Clostridium perfringens has bent DNA in a promoter region. It has been shown to possess another weakly-bent DNA in the coding region, which regulates epsilon-toxin gene expression along with the upstream bent DNA. When a DNA fragment coveting the two bent DNA regions were PCR amplified and cloned into an E coli plasmid, A-tract consisting of 8 adenine residues located at the downstream bent DNA lost the 8th adenine. This suggests that cloning of the fragment causes plasmid instability. We constructed plasmids expressing C. perfringens LrpC with and without His' by using our plasmid vector pFF. Unfortunately, transformats of C. perfringens strains 13 and SM101 carrying these plasmids failed to produce large amounts of recombinant LrpC proteins. Therefore, we failed to purify LrpC from these cultures. One likely reason for the difficulty of purification is possible proteolytic breakdown of the recombinant product during purification. We constructed a clostripain- … More like protease-deficient mutant, since this is the most potent thiol-protease among proteases produced by the organism and probably decreases the yield of LrpC products. We are currently attempting to purify LrpC by using this mutant To assess whether or not his-tagged LrpC functions normally, we examined biological properties of both transformants carrying plasmids containing LrpC and LrpC-his genes. The result indicates LrpC and LrpC-his are involved in the onset of spore-formation, as demonstrated for Bacillus subtilis, proving that LrpC-his is fractional in C perfringens. We prepared cell lysate from Strain 13 with and without a plasmid containing the LrpC gene and also PCR-products corresponding to the fragment containing the two bent DNA regions of the epsilon-toxin gene. The gel retardation assay using these did not show the specific interaction between the DNA and LrpC probably because of impurity of protein samples or the presence of LrpC in the wild type as well as in the transformant. To solve theseproblems, we are currently purifying LrpC-his by using Ni-chelating Sepharose from large-scale culture of the C. perfringens transformant We have also attempted an alternative method involving an E coli expression system as reported for purification of B. subtilis LrpC. Since we constructed a LrpC gene-disrupted mutant, we have undertaken the gel retardation assay by using lysates from a wild-type strain and its isogenic LrpC(-)mntant. Less

灌注梭状芽胞杆菌的埃氏蛋白酶毒素基因在启动子区域中具有BEN DNA。已显示它在编码区中具有另一个弱弯曲的DNA，该DNA与上游弯曲DNA一起调节了埃氏蛋白石毒素基因的表达。当DNA碎片垂涎的两个弯曲DNA区域被PCR扩增并克隆到一个大肠杆菌质粒中时，由位于下游Bento DNA的8个腺嘌呤保留量组成的A诱饵损失了第8腺嘌呤。这表明片段的克隆会导致质粒不稳定。我们通过使用我们的质粒矢量PFF构建了表达浓度杆菌LRPC的质粒。不幸的是，携带这些质粒的C. perfringens菌株13和SM101的转化未能产生大量的重组LRPC蛋白。因此，我们未能从这些文化中净化LRPC。纯化难度的可能原因是我们构建了一个闭合 - 更像是蛋白酶缺陷的突变体，因为这是生物体产生的蛋白酶中最有效的硫醇 - 蛋白酶，并且可能会降低LRPC产品的产量。我们目前正在尝试通过使用该突变体来评估HIS标记的LRPC是否正常功能来纯化LRPC，我们检查了携带含有LRPC和LRPC基因的两种转化体的生物学特性。结果表明，LRPC和LRPC-HIS参与了酱油形成的发作，如枯草芽孢杆菌所证明的那样，只要LRPC-HIS在c灌注中是分数。我们制备了菌株13的细胞裂解物，有无质粒含有LRPC基因的质粒，还制备了与含有Epsilon-Toxin基因的两个弯曲DNA区域的片段相对应的PCR产物。使用它们的凝胶延迟测定未显示DNA和LRPC之间的特定相互作用，这可能是由于蛋白质样品的杂质或野生型和转化剂中LRPC的存在。为了解决这些问题，我们目前正在通过使用C. perprringens转化物的大规模培养物中使用Ni-CheLPC-HIS纯化LRPC-HIS，我们还尝试了一种涉及E Coli表达系统的替代方法，据报道，用于纯化枯草芽孢杆菌LRPC。由于我们构建了一个LRPC基因 - 脱离的突变体，因此我们通过使用野生型菌株的裂解物及其同源性LRPC（ - ）Mntant进行了凝胶延迟测定。较少的