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首页> 外文期刊>Applied Microbiology >A Synthetic Arabinose-Inducible Promoter Confers High Levels of Recombinant Protein Expression in Hyperthermophilic Archaeon Sulfolobus islandicus
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A Synthetic Arabinose-Inducible Promoter Confers High Levels of Recombinant Protein Expression in Hyperthermophilic Archaeon Sulfolobus islandicus

机译：合成的阿拉伯糖诱导型启动子赋予高水平嗜热古细菌Sulfolobus islandicus中高水平的重组蛋白表达。

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Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus , a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P_( araS -SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P_( araS -SD) directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N- lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N- lacS . These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.
机译：尽管在超嗜热古细菌的遗传研究中取得了重大进展，但这些生物中重组蛋白的生产始终遭受着低产量的困扰，并且仍然需要强大的表达系统。在这里，我们报告了一种多用途的载体，可赋予其在嗜热性鱼眼的Sulfolobus islandicus中高水平表达蛋白质的能力。构建了两个表达载体pSeSD和pEXA，它们具有11个独特的限制性位点。它们包含两个六组氨酸（6×His）肽标签的编码序列，以及编码两个蛋白酶位点的编码序列，后者使从表达的重组蛋白中去除肽标签成为可能。虽然pEXA使用araS启动子进行蛋白表达，但pSeSD使用P_（araS -SD），这是一种带有工程核糖体结合位点（RBS; Shine-Dalgarno [SD]序列）的araS衍生物启动子。我们发现P_（araS -SD）指导高水平的靶基因表达。更令人惊讶的是，重组蛋白的N端氨基酸测序揭示了从pEXA-N-lacS合成的蛋白缺乏设计的6×His标签，并且翻译起始不是从融合基因的ATG密码子开始的。相反，它始于6×His密码子下游的多个位点。有趣的是，在ATG密码子上游插入一个RBS位点可重新获得6xHis标签的表达，如pSeSD-N-lacS所示。这些结果产生了对古细菌翻译机制的新颖见解。 Crearchaeon硫磺菌可以利用蛋白质的N末端编码序列来指定在没有RBS位点的情况下的翻译起始。

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