Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.
Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.
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DOI：
10.1186/1472-6750-6-13
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发表时间：
2006-03-06
影响因子：
3.5
通讯作者：
Morin GB
中科院分区：
文献类型：
--
作者：
Colwill K;Wells CD;Elder K;Goudreault M;Hersi K;Kulkarni S;Hardy WR;Pawson T;Morin GB
已经开发了重组系统来快速将打开的阅读框（ORF）迅速穿梭到多个表达矢量中，以分析后基因组时代可用的大量CDNA。在创建者系统中，可以将引入供体向量的ORF用CRE重组酶转移到针对不同应用优化的受体向量库中。创建器系统的可用性受到轻松操纵DNA的能力，下游应用的受体向量的数量以及创建器向量的蛋白质表达水平。
迄今为止，我们已经开发了20多种新型受体矢量，这些媒介采用了通常用于蛋白质组学应用和基因功能分析的各种启动子和表位标签。我们还对供体向量进行了多种增强功能，包括添加不同的多个克隆位点，以允许从预先存在的矢量穿梭和引入LACZ Alpha报告基因以进行选择。重要的是，为了改善对5'TAG和ORF之间LOXP位点蛋白质表达的任何影响，我们将剪接事件引入了我们的表达矢量。由结果的“创建者拼接”向量产生的消息在哺乳动物系统中经历剪接以删除LOXP站点。通过分析创建者的剪接构建体，我们发现蛋白质表达水平也显着提高。
在克隆过程中，新的供体和受体矢量的开发增强了多功能性，并使该系统与多种多样的下游应用兼容。我们的创建者剪接系统中引入的修改旨在消除由于重组而导致的无关序列，但也通过增加蛋白质表达水平来帮助下游分析。结果，我们现在可以采用效率较低的表位标签，并降低我们的测定量表以允许更高的吞吐量。创建者剪接系统似乎是蛋白质组学的极有用的工具。
Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.
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影响因子：
3.2
作者：
GAY, P;LECOQ, D;HOCH, JA
通讯作者：
HOCH, JA
影响因子：
9.2
作者：
Liu, QH;Li, MZ;Elledge, SJ
通讯作者：
Elledge, SJ
影响因子：
11.4
作者：
Le Hir, H;Izaurralde, E;Moore, MJ
通讯作者：
Moore, MJ
影响因子：
7.7
作者：
Bourdon, V;Harvey, A;Lonsdale, DM
通讯作者：
Lonsdale, DM
DOI：
10.1073/pnas.76.9.4317
发表时间：
1979-01-01
影响因子：
11.1
作者：
GRUSS, P;LAI, CJ;KHOURY, G
通讯作者：
KHOURY, G