High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins
High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins
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DOI：
10.1021/pr700856s
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发表时间：
2008-05-01
影响因子：
4.4
通讯作者：
Kim, Dong-Myung
中科院分区：
文献类型：
--
作者：
Ahn, Jin-Ho;Keum, Jung-Won;Kim, Dong-Myung
我们描述了一种高通量策略，通过工程重组蛋白的早期核苷酸序列来调节重组蛋白的表达。将靶基因的+2和+3密码子随机化后，在体内分离每个变异基因，随后使用体外蛋白合成技术表达。当数百个克隆被并行检测时，发现目标基因的表达水平根据随机区域密码子的身份变化高达70倍。这种广泛而连续的表达水平分布使得选择特定的密码子排列来表达目标基因达到所需的水平。此外，当相同的基因在体内表达时，蛋白质表达的密码子依赖性变异也会重现。因此，我们期望本文报道的方法可以作为在体外或体内调节水平下快速表达蛋白质分子的通用平台。
We describe a high-throughput strategy for tuning the expression of recombinant proteins through engineering their early nucleotide sequences. After randomizing the +2 and +3 codons of the target genes, each of the variant genes was isolated in vivo and subsequently expressed using ill vitro protein synthesis techniques. When several hundreds of clones were examined in parallel, it was found that expression levels of target genes varied as much as 70-fold depending on the identity of the codons in the randomized region. This broad and continuous distribution of expression levels enabled the selection of specific codon arrangements for the expression of target genes at a desired level. Furthermore, codon-dependent variations in protein expression were reproduced when the same genes were expressed in vivo. Thus, we expect that the methodology reported here could be utilized as a versatile platform for rapid expression of protein molecules at modulated levels either in vitro or in vivo.