Structure and function of halotolerant metalloprotein from extremely halophilic archaeon

日本海葵菌株TR-1是一个主要的三角形圆盘状，极端嗜盐的总执事。我们从Ha中纯化了一种新的铁氧还蛋白（Fd）。用阴离子交换色谱法和凝胶过滤法测定粳稻的电泳均匀性。sds -聚丙烯酰胺凝胶电泳显示其表观分子质量为24 kDa。氨基酸组成分析表明，Ha。粳稻Fd含有许多酸性氨基酸。n端氨基酸序列为H2N-PTV-EYLNYEVVDDNGWDMYDDDVFAEASDM。Ha的吸收光谱和ESR光谱。japonica Fd显示Fd含有一个[2Fe-2S]簇。利用Ha基因组DNA进行聚合酶链反应（PCR）。以粳稻为模板。该引物是根据Ha的n端氨基酸序列设计的。粳稻；反义引物是基于其他盐菌Fds中保守的多肽区。该DNA片段长约160 bp，推测为Fd基因的一部分，通过PCR扩增得到，并用作基因组DNA Southern杂交分析的探针。用Sal I酶切的基因组DNA显示一个3.1 kb的杂交带。构建了一个包含约3 kb Sal I消化片段的亚基因组文库，并通过集落杂交筛选Fd基因。获得了一个阳性克隆，发现含有一个3.1 kb的Sal I插入的重组质粒。该3.1 kb片段的核苷酸序列显示一个387 nt的开放阅读框，编码129个氨基酸的多肽。在假设对初始蛋氨酸进行加工的情况下，推导出的n端氨基酸序列与肽段测序结果一致。氨基酸序列与菠菜、marismortui Haloarcula marismortui和salinarium Fds的同源性分别为43%、98%和87%。来自哈的Fds。与菠菜Fd相比，粳稻和其他盐菌在n端区域多出20个a.a.。这个n端区域似乎对耐盐性起着重要作用。接下来，哈。将粳稻Fd基因连接到表达质粒pET-2[d(*)]和pGEX-5X-1上，转化到大肠杆菌中。重组Fds与t7标签或谷胱甘肽5-转移酶成功融合。然而，两种Fd融合都不包含Fe-S簇。少

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