Structure and function of halotolerant metalloprotein from extremely halophilic archaeon

极嗜盐古菌耐盐金属蛋白的结构和功能

• 批准号: 09440228
• 负责人: NAKAMURA Satoshi
• 金额: $ 8.38万
• 依托单位: TOKYO INSTITUTE OF TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09440228/
• 关键词: Ferredoxin; Extremely halophilic archaeon; Purification; Fe-S cluster; PCR; Gene cloning; Fusion expression

Halaarcula japonica strain TR-1 is a predominantly triangular disk-shaped, extremely halophilic archdeacon. We have purified a novel ferredoxin (Fd) from Ha. japonica to electrophoretic homogeneity by anion exchange chromatography and gel filtration. It had an apparent molecular mass of 24 kDa on SDS-polyacrylamide gel electrophoresis. Amino acid composition analysis revealed that the Ha. japonica Fd contained many acidic amino acids. The N-terminal amino acid sequence was determined to be H2N-PTV-EYLNYEVVDDNGWDMYDDDVFAEASDM.The absorption and ESR spectra of the Ha. japonica Fd revealed that the Fd contained a [2Fe-2S] cluster. Polymerase chain reaction (PCR) was performed using the genomic DNA of Ha. japonica as a template. The sense primer was designed from the N-terminal amino acid sequence of the Ha. japonica Fd ; the antisense primer was based on the conserved polypeptide region among other halobacterial Fds. The DNA fragment of about 160 bp, presumed to be a part of the Fd gene, … More was amplified by PCR and used as a probe in Southern hybridization analyses of genomic DNA.Genomic DNA digested with Sal I showed a hybridization band at 3.1 kb. A subgenomic library, I containing Sal I digests of about 3 kb, was constructed and screened for the Fd gene by colony hybridization. A positive clone was obtained and Found to contain a recombinant plasmid with a 3.1-kb Sal I insert. The nucleotide sequence of the 3.1-kb fragment revealed an open reading frame of 387 nt coding for a polypeptide of 129 amino acids. The deduced amino acid sequence at the N-terminal region is in agreement with that determined by peptide sequencing, assuming a processing the initial methionine. The amino acid sequence showed about 43%, 98% and 87% identities with those of spinach, Haloarcula marismortui and Halobacterium salinarium Fds, respectively. Fds from Ha. Japonica and other halobacteria had an additional 20 a.a. in the N-terminal region compared to the spinach Fd. This N-terminal region seemed to play an important role for halotolerance. Next, the Ha. japonica Fd gene was ligated to the expression plasmids pET-2[d(*) and pGEX-5X-1, and then transformed to Escharichia coli. The recombinant Fds were successfully produced as fusion proteins with T7tag or glutathione 5-transferase. Both Fd fusions, however, did not contain Fe-S clusters. Less

Halaarcula jizonica菌株tr-1是一种以三角形盘状为主的极端嗜盐性的执事。我们从Ha中提纯了一个新的铁氧还蛋白(Fd)。采用阴离子交换层析和凝胶过滤的方法，获得了均一的电泳谱。经SDS-聚丙烯酰胺凝胶电泳法测定，其表观分子量为24 kDa。氨基酸组成分析表明，Ha。海带中含有多种酸性氨基酸。N-末端氨基酸序列为H_2N-PTV-EYLNYEVDDNGWDMYDDDVFAEASM。结果表明，该Fd含有一个[2Fe-2S]簇。用Ha的基因组DNA进行聚合酶链式反应(PCR)。以粳稻为模板。根据Ha的N-端氨基酸序列设计了正义引物。该反义引物基于盐生细菌FDS中保守的多肽区。约160bp的DNA片段，推测为Fd基因…的一部分对基因组DNA进行Southern杂交分析，Sal I酶切后的基因组DNA在3.1kb处出现一条杂交条带。构建了含有约3kb Sal I酶切片段的亚基因组文库I，并通过菌落杂交筛选了Fd基因。获得了一个阳性克隆，该克隆含有一个带有3.1kb Sal I插入片段的重组质粒。该3.1-kb片段的核苷酸序列显示了一个387nT的开放阅读框，编码129个氨基酸的多肽。推定的N-末端氨基酸序列与多肽测序结果一致，假定以蛋氨酸为起始氨基酸进行加工。该序列与菠菜、盐生盐藻和盐生盐生杆菌的氨基酸序列同源性分别为43%、98%和87%。来自哈州的FDS。日本和其他盐生细菌有额外的20A.A.在N-末端区域与菠菜FD相比。这一N-末端区域似乎对耐盐性起着重要作用。接下来是哈。将粳稻Fd基因连接到表达载体pET-2[d(*)]和pGEX-5X-1上，转化大肠杆菌。成功地将重组FDS与T7tag或谷胱甘肽5-转移酶融合表达。然而，这两个FD融合蛋白都不包含Fe-S簇。较少

项目成果

Daisuke Sugimori: "Purification and characterization of a ferredoxin from Haloareula japonica strain TR-1, and cloning of its gene" Journal of Inorganic Chemistry. 67. 250-250 (1997)

Daisuke Sugimori：“来自 Haloareula japonica 菌株 TR-1 的铁氧还蛋白的纯化和表征，及其基因的克隆”《无机化学杂志》。

D.Sugimori 他: "Purification and Characterization of a ferredoxin from Haloarcula japonica strain TR-1,and cloning of its gene" J.Inorg Chem.67. 250 (1997)

D. Sugimori 等人：“来自 Haloarcula japonica 菌株 TR-1 的铁氧还蛋白的纯化和表征，及其基因的克隆”J. Inorg Chem.67 (1997)。

Ryousuke Mizui: "Amino Acid Substitutions at the Subunit Interface Cause Thermal Stabilization in IPMDH of Bacillus coagulans" Nucleic Acids Symposium Series. 39. 203-204 (1998)

Ryousuke Mizui：“亚基界面的氨基酸取代导致凝结芽孢杆菌 IPMDH 的热稳定性”核酸研讨会系列。

Akiko Ikeda: "Molecular cloning of the gene encoding a 2Fe-2S ferredoxin from extremely holophilic archaeon Haloarcula japonica strain TR-1" Nucleic Acids Symposium Series. 37. 109-110 (1997)

Akiko Ikeda：“从极其亲全的古菌 Haloarcula japonica 菌株 TR-1 中编码 2Fe-2S 铁氧还蛋白的基因的分子克隆”核酸研讨会系列。

Akiko Sugio: "Genetics, Biochemistry and Ecology of Cellulose Degradation" Uni Publisher 印刷中, (1999)

Akiko Sugio：《纤维素降解的遗传学、生物化学和生态学》Uni Publisher 已出版，（1999 年）