Modulation of ColE1-like plasmid replication for recombinant gene expression.
Modulation of ColE1-like plasmid replication for recombinant gene expression.
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DOI：
10.2174/187221510790410822
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发表时间：
2010-01
期刊：
影响因子：
--
通讯作者：
Camps M
中科院分区：
文献类型：
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作者：
Camps M
ColE 1样质粒构成了最常用的重组蛋白表达载体。ColE 1质粒复制受反义RNA机制的严格控制，该机制是高度动态的，将质粒代谢负荷调节到宿主的生理状态。由于非生理水平的表达和重组蛋白质的经常偏向的氨基酸组成，在诱导重组蛋白质表达时质粒稳态被打乱。质粒复制的失调是基于质粒的表达系统崩溃的主要原因，因为代谢负荷（由于平均拷贝数增加）和产生无质粒细胞的概率（由于拷贝数变异增加）同时增加。共存质粒的调节元件之间的干扰对质粒稳定性产生相当的影响（质粒不相容性）。调节重组基因表达的质粒拷贝数旨在实现高基因剂量，同时保持表达系统的稳定性。在这里，我提出了针对质粒复制优化重组基因表达的策略。具体而言，我审查的方法，旨在调节反义调控系统（以及它们的影响质粒不相容性）和创新的策略，涉及调制宿主因子，R-环的形成，和重组基因表达的时间。
ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of R-loop formation, and of the timing of recombinant gene expression.