Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase.
Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase.
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DOI：
10.1002/bit.22491
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发表时间：
2009-12-15
影响因子：
3.8
通讯作者：
Forster, Anthony C.
中科院分区：
文献类型：
--
作者：
Du, Liping;Gao, Rong;Forster, Anthony C.
在体外或体内工程蛋白表达通常是简单的单基因，但仍然具有挑战性的多个基因，因为需要协调控制。RNA和蛋白质过表达策略通常利用T7 RNA聚合酶及其天然TΦ I类终止子。然而，这种终止子的效率低和大尺寸（100 bp）对于多基因构建和表达是有问题的。在这里，我们测量的串联拷贝的小（18 bp）II类T7终止子水泡性口炎病毒在体外转录和翻译在体外和体内的影响。我们首先测试单体和二聚体基因构建体，然后尝试延伸到五聚体基因构建体。构建pET载体和翻译因子基因的“BioBrick”版本以促进克隆，并掺入His-标签以允许共纯化所有蛋白质产物以进行相对无偏倚的分析和容易的纯化。几个结果是令人惊讶的，包括五聚体构建体在体内的不平衡表达，说明了合成生物学研究基因表达的价值。然而，通过改变基因的顺序和向上游基因添加额外的启动子或转移到更可预测的体外翻译系统，这些问题得到了合理的解决。考虑到我们最初的意外结果，这些成功是重要的，并且我们不知道五种蛋白质协调过表达的另一个例子。我们的模块化，灵活，合理的方法应该进一步授权合成生物学家希望同时过表达多种蛋白质。
Engineering protein expression in vitro or in vivo is usually straightforward for single genes, but remains challenging for multiple genes because of the requirement of coordinated control. RNA and protein overexpression strategies often exploit T7 RNA polymerase and its natural TΦ Class I terminator. However, this terminator’s inefficiency and large size (100 bp) are problematic for multigene construction and expression. Here, we measure the effects of tandem copies of a small (18 bp) Class II T7 terminator from vesicular stomatitis virus on transcription in vitro and on translation in vitro and in vivo. We first test monomeric and dimeric gene constructs, then attempt extension to pentameric gene constructs. “BioBrick” versions of a pET vector and translation factor genes were constructed to facilitate cloning, and His-tags were incorporated to allow copurification of all protein products for relatively unbiased analysis and easy purification. Several results were surprising, including imbalanced expression of the pentameric constructs in vivo, illustrating the value of synthetic biology for investigating gene expression. However, these problems were solved rationally by changing the orders of the genes and by adding extra promoters to the upstream gene or by moving to a more predictable in vitro translation system. These successes were significant, given our initial unexpected results and that we are unaware of another example of coordinated overexpression of five proteins. Our modular, flexible, rational method should further empower synthetic biologists wishing to overexpress multiple proteins simultaneously.
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