Design parameters to control synthetic gene expression in Escherichia coli.
Design parameters to control synthetic gene expression in Escherichia coli.
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DOI：
10.1371/journal.pone.0007002
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发表时间：
2009-09-14
期刊：
影响因子：
3.7
通讯作者：
Gustafsson C
中科院分区：
文献类型：
--
作者：
Welch M;Govindarajan S;Ness JE;Villalobos A;Gurney A;Minshull J;Gustafsson C
作为治疗剂、研究试剂和分子工具的蛋白质的生产通常取决于在异源宿主中的表达。合成基因越来越多地用于蛋白质生产，因为序列信息比相应的物理DNA更容易获得。蛋白质编码序列通常被重新设计以增强表达，但没有实验支持的设计原则。为了鉴定影响蛋白表达的序列特征，我们合成并在E.大肠杆菌两套40个基因编码两个商业价值的蛋白质，DNA聚合酶和单链抗体。不同的基因只在同义密码子的使用表达蛋白质的水平范围从检测不到的细胞蛋白质的30%。使用偏最小二乘回归，我们测试了蛋白质生产水平与已报道影响表达的参数的相关性。我们发现E.大肠杆菌中的氨基酸序列高度依赖于用于编码氨基酸子集的密码子。有利的密码子主要是那些在氨基酸饥饿期间被最高电荷的tRNA读取的密码子，而不是在高表达的大肠杆菌中最丰富的密码子。coli蛋白。最后，我们通过使用预测表现良好的密码子偏好设计、合成和测试新基因来证实我们的模型的有效性。在这项研究中，基因设计参数的系统分析，使我们能够确定密码子使用的基因作为一个关键的决定因素，可实现的蛋白质表达水平在大肠杆菌。杆菌我们为此提出了一个生物化学基础，以及设计算法，以确保从合成基因中产生高蛋白质。这种方法的复制应允许类似的设计算法，以经验得出的任何表达系统。
Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles. To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well. The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system.
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影响因子：
1.6
作者：
Fuglsang, A
通讯作者：
Fuglsang, A
DOI：
10.1073/pnas.86.10.3699
发表时间：
1989-05-01
影响因子：
11.1
作者：
GUTMAN, GA;HATFIELD, GW
通讯作者：
HATFIELD, GW
影响因子：
3.2
作者：
DONG, HJ;NILSSON, L;KURLAND, CG
通讯作者：
KURLAND, CG
影响因子：
4.1
作者：
Harcum, SW
通讯作者：
Harcum, SW
影响因子：
7.7
作者：
Dittmar, KA;Sorensen, MA;Pan, T
通讯作者：
Pan, T