PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome
PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome
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DOI：
10.1093/protein/12.12.1113
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发表时间：
1999-12-01
期刊：
影响因子：
--
通讯作者：
Blackman, MJ
中科院分区：
文献类型：
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作者：
Withers-Martinez, C;Carpenter, EP;Blackman, MJ
人类疟原虫恶性疟原虫（Plasmodium falciparum）富含A+ t的基因组编码具有重要生物学意义的基因，这些基因在异源真核系统中无法有效表达，这是由于密码子的使用极度偏倚和大量隐聚腺苷化位点的存在。在这项工作中，我们优化了一种组装聚合酶链反应（PCR）方法，用于快速和极准确地合成2.1 kb的恶性疟原虫编码枯草杆菌样蛋白酶的基因（pfsub-1）。利用计算机软件设计了104个寡核苷酸，通过单步PCR对其进行了组装，并通过PCR扩增得到了一个合成基因，该基因在毕赤酵母和重组杆状病毒感染的High Five（TM）细胞中均成功表达。我们相信这种策略是特别有趣的，因为它简单，易于获取，并且不受要合成的基因大小的限制，作为疟疾基因组或任何其他富含a + t的生物体的系统方法，该方法允许快速合成核苷酸序列，优化其在选择系统中的表达，并生产足够大数量的生物材料，以完成分子和结构表征。
The A+T-rich genome of the human malaria parasite Plasmodium falciparum encodes genes of biological importance that cannot be expressed efficiently in heterologous eukaryotic systems, owing to an extremely biased codon usage and the presence of numerous cryptic polyadenylation sites. In this work we have optimized an assembly polymerase chain reaction (PCR) method for the fast and extremely accurate synthesis of a 2.1 kb Plasmodium falciparum gene (pfsub-1) encoding a subtilisin-like protease. A total of 104 oligonucleotides, designed with the aid of dedicated computer software, were assembled in a single-step PCR, The assembly was then further amplified by PCR to produce a synthetic gene which has been cloned and successfully expressed in both Pichia pastoris and recombinant baculovirus-infected High Five(TM) cells. We believe this strategy to be of special interest as it is simple, accessible and has no limitation with respect to the size of the gene to be synthesized, Used as a systematic approach for the malarial genome or any other A + T-rich organism, the method allows the rapid synthesis of a nucleotide sequence optimized for expression in the system of choice and production of sufficiently large amounts of biological material for complete molecular and structural characterization.