High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system
High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system
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DOI：
10.1016/j.pep.2005.11.007
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发表时间：
2006-06-01
影响因子：
1.6
通讯作者：
Fox, Brian G.
中科院分区：
文献类型：
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作者：
Blommel, Paul G.;Martin, Peter A.;Fox, Brian G.
结构基因组学和蛋白质组学倡议的成功取决于适合于蛋白质生产的载体中靶基因的可用性。在这里，我们比较了两种高通量的方法，从质粒衍生的cDNA片段生产表达载体。构建表达载体以与Gateway重组克隆系统和基于Flexi Vector限制的克隆系统兼容。通过产生序列验证的表达克隆，对来自PCR的96个不同靶基因平行进行每个系统的克隆方案。为Flexi Vector克隆准备目标开放阅读框所需的短核苷酸序列允许一步PCR方案，从而相对于Gateway方案产生更少的突变。此外，通过将靶开放阅读框直接初始克隆到表达载体中，与Gateway系统所需的方案相比，Flexi Vector系统节省了时间和成本。在Flexi Vector系统中，基因在四种不同的表达载体之间转移。Flexi载体之间的基因转移效率取决于包括与限制性位点之一相邻的序列同一性区域。在载体的侧翼序列中适当构建的情况下，证明了95-98%的基因转移效率。(c)2005年爱思唯尔公司All rights reserved.
The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway recombination cloning system and the Flexi Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi Vector system gave time and cost savings compared to the protocol required for the Gateway system. Within the Flexi Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were demonstrated. (c) 2005 Elsevier Inc. All rights reserved.