Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.
Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.
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DOI：
10.1016/j.ijantimicag.2006.08.029
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发表时间：
2007
影响因子：
10.8
通讯作者：
T. K. Miller;C. Patel;C. Selitrennikoff
中科院分区：
文献类型：
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作者：
T. K. Miller;C. Patel;C. Selitrennikoff
核苷水解酶（NH）（EC 3.2.2.3）是许多原生动物寄生虫利用的嘌呤-嘧啶补救途径中的必需酶，并且可能是有用的药物靶标。然而，NH抑制剂的研究受到缺乏合适的体外筛选的阻碍。我们已经构建了一个酿酒酵母菌株，需要表达的利什曼原虫主要核苷水解酶（LmNH）酶的增长，并可能适合作为NH抑制剂的筛选。用L.主要基因组DNA作为模板，克隆到表达载体中，并用于转化不能在尿苷作为尿嘧啶的唯一来源上生长的酵母突变体。LmNH的表达产生了ca. 35.6 kDa蛋白，其被证明是功能性的，因为突变菌株能够在含有尿苷作为尿嘧啶的唯一来源的培养基上生长。重要的是，这项工作已经产生了一种菌株，可用于筛选作为这种必需利什曼原虫酶的潜在抑制剂的化合物。
Nucleoside hydrolase (NH) (EC 3.2.2.3) is an essential enzyme in the purine–pyrimidine salvage pathway utilised by many protozoan parasites and may be a useful drug target. However, the search for NH inhibitors has been hampered by the lack of suitable in vitro screens. We have constructed a Saccharomyces cerevisiae strain that requires expression of the Leishmania major nucleoside hydrolase (LmNH) enzyme for growth and that may be suitable as a screen for NH inhibitors. The gene encoding LmNH was amplified using polymerase chain reaction with L. major genomic DNA as the template, cloned into an expression vector and used to transform a yeast mutant unable to grow on uridine as the sole source of uracil. Expression of LmNH yielded an ca. 35.6kDa protein, which was shown to be functional as the mutant strain was able to grow on medium containing uridine as the sole source of uracil. Importantly, this work has resulted in a strain that can be used to screen compounds as potential inhibitors of this essential Leishmania enzyme.