Department of Biomedical Science, Cheongju University, Cheongju 28160, Republic of Korea.
College of Pharmacy, Duksung Women's University, Seoul 01369, Republic of Korea.
Int J Mol Sci. 2023 Jul 4;24(13):11083. doi: 10.3390/ijms241311083.
is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this Rosetta(DE3), containing the original gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by .
是疟疾最广泛的原因，特别是在亚太和美洲等亚热带和温带地区。乳酸脱氢酶（PvLDH）是糖酵解途径中的一种必需酶，对于寄生虫的发育和繁殖是必需的。因此，这些寄生虫的 LDH 作为疟疾的诊断生物标志物和开发抗疟药物的潜在分子靶标引起了关注。在这项研究中，我们制备了一种未经密码子优化的 PvLDH 过表达转化菌株。我们将插入 基因的重组质粒 DNA 插入 pET-21a(+)表达载体中，导入适合真核蛋白表达的 Rosetta(DE3)菌株。优化了 PvLDH 从含有原始 基因的 Rosetta(DE3)表达的时间、温度和诱导剂浓度。我们从 Rosetta(DE3)菌株中获得了 31.0mg/L 的 PvLDH 产量和>95%的高纯度。对纯化的蛋白质进行了结构和功能表征。从没有密码子优化的转化细菌中表达和纯化的 PvLDH 成功地证明了其潜在的四聚体结构和酶活性。这些发现有望为研究传染病、代谢、疟疾的诊断和治疗提供有价值的见解。
