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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Isolation of a low-molecular-weight, multicopy plasmid, pNHK101, from Thermus sp TK10 and its use as an expression vector for T-thermophilus HB27
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Isolation of a low-molecular-weight, multicopy plasmid, pNHK101, from Thermus sp TK10 and its use as an expression vector for T-thermophilus HB27

机译：Thermus sp TK10的低分子量多拷贝质粒pNHK101的分离及其作为T-嗜热菌HB27表达载体的用途

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摘要

We isolated a small multicopy cryptic plasmid, pNHK101, from Thermus sp. TK10 for use as a replicon of a Thermus expression vector. The nucleotide sequence of pNHK101 revealed that this plasmid was 1564 bp long, with a total G+C content of 66.8%, which was in agreement with that of Thermus genomic DNA. The sequence did not show any significant similarities to any other plasmids; also, the amino acid sequences of four putative open reading frames, found in the plasmid, did not show strong similarities to those in the databases, except the ORFI, which had very slight similarities to several replication proteins of plasmids from other bacteria. pNHK101 was able to replicate in Thermus thermophilus HB27 with copy number about 80, and was stably maintained at 60 degrees C, but became unstable at 70 degrees C. Based on pNHK101, we constructed a plasmid vector, pKMH052, containing the highly thermostable kanamycin resistance gene as a selective marker. The copy number of pKMH052 decreased to about one-fourth of that of pNHK101, but stability at 60 degrees C did not alter under non-selective conditions. pKMH052 was compatible with pTT8, and interestingly, the presence of pTT8 in the same cells improved the stability of pKMH052 at 70 degrees C. Cloning of the crtB gene of T thermophilus HB27 encoding phytoene synthase into pKMH052, and introduction into T thermophilus cells resulted in a 2.8-fold production of carotenoids, indicating the potential use of this plasmid for overexpression of genes from thermophiles and hyperthermophiles. (c) 2005 Elsevier Inc. All rights reserved.
机译：我们从Thermus sp。中分离出了一个小的多拷贝隐性质粒pNHK101。 TK10用作Thermus表达载体的复制子。 pNHK101的核苷酸序列显示该质粒长1564 bp，总G + C含量为66.8％，与Thermus基因组DNA一致。该序列与任何其他质粒均未显示任何显着相似性。同样，在质粒中发现的四个假定的开放阅读框的氨基酸序列与数据库中的氨基酸序列没有显示出强烈的相似性，除了ORFI，其与来自其他细菌的质粒的几种复制蛋白非常相似。 pNHK101能够在拷贝数为80的嗜热栖热菌HB27中复制，并稳定地维持在60摄氏度，但在70摄氏度时变得不稳定。基于pNHK101，我们构建了具有高度热稳定性卡那霉素抗性的质粒载体pKMH052基因作为选择标记。 pKMH052的拷贝数降低到pNHK101的约四分之一，但在非选择性条件下在60摄氏度下的稳定性没有改变。 pKMH052与pTT8相容，有趣的是，同一细胞中pTT8的存在提高了pKMH052在70摄氏度时的稳定性。将嗜热性粒细胞HB27的植物植酸合酶的crtB基因克隆到pKMH052中，并引入到嗜热性粒细胞中类胡萝卜素的产量为2.8倍，表明该质粒可用于从嗜热菌和嗜热菌中过度表达基因。 （c）2005 Elsevier Inc.保留所有权利。

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