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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis
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Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

机译：乳酸克鲁维酵母LAC4启动子变异体在细菌中缺乏功能但在乳酸克鲁维酵母中仍具有全部功能

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The strong LAC4 promoter (PLAC4) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of PLAC4 to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of PLAC4 variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with PLAC4. Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ∼87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type PLAC4. We created a novel integrative expression vector (pKLAC1) containing the PLAC4 variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.
机译：来自乳酸克鲁维酵母的强LAC4启动子（PLAC4）已被广泛用于驱动这种在工业上重要的酵母中异源蛋白的表达。这种表达方法的一个缺点是PLAC4在大肠杆菌中具有促进基因表达的偶然能力。这可能会干扰在将表达构建体引入酵母细胞之前在大肠杆菌细胞中组装表达构建体的过程，尤其是如果克隆的基因编码对细菌有害的蛋白质时尤其如此。在这项研究中，我们通过对三个DNA序列（PBI，PBII和PBIII）进行定向诱变，创建了一系列PLAC4变体，这些序列类似于细菌启动子的大肠杆菌Pribnow盒元件，并且位于两个大肠杆菌转录起始上游与PLAC4相关的网站。 PBI的突变使报告蛋白（绿色荧光蛋白[GFP]）的细菌表达降低了约87％，而PBII和PBIII的突变对GFP的表达影响很小。全部三个序列的删除完全消除了GFP表达。此外，每个启动子变体在乳酸克鲁维酵母细胞中表达人血清白蛋白的水平可与野生型PLAC4相当。我们创建了一个新型的整合表达载体（pKLAC1），其中包含缺少PBI的PLAC4变体，并使用它成功克隆并表达了牛肠激酶的催化亚基，该酶在大肠杆菌细胞中历来是有问题的。 pKLAC1载体应先在大肠杆菌中克隆其他潜在毒性基因，然后再在乳酸克鲁维酵母中表达。

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