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首页> 外文期刊>Biotechnology and Bioengineering >Enhancing Functional Expression of Codon-Optimized Heterologous Enzymes in Escherichia coli BL21(DE3) by Selective Introduction of Synonymous Rare Codons
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Enhancing Functional Expression of Codon-Optimized Heterologous Enzymes in Escherichia coli BL21(DE3) by Selective Introduction of Synonymous Rare Codons

机译：通过选择性引入同义稀有密码子来增强CONCHERICA COLI BL21（DE3）中的密码子优化异源酶的功能表达

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摘要

Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 30 end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. (C) 2016 Wiley Periodicals, Inc.
机译：异源基因中的稀有密码子可能导致蛋白质合成的过早终止，氨基酸的MISINC丙烷和/或mRNA的慢速翻译，降低异源蛋白表达。然而，其具有与功能性蛋白质折叠有关的假设功能已经几乎没有报道。在这里，我们研究了从热氨基乙酰丙乳溶液和酰氨酸碱基来自Hertreptomyces Caatingaensis的热氨基杆菌和酰胺水解酶基因的两种密码子（IE，稀有囊蒙）基因蔗糖磷酸化酶（SP）基因的影响。大肠杆菌大肠杆菌BL21（DE3）。我们调查了在罕见的密码子SP和AH基因的前半部分中引入α-螺旋，β-链或接头的编码区域。与其他区域相比，在β-链的编码区域开始时引入单个SRC的单个SRC大大提高了它们的可溶性表达水平。此外，我们应用了针对含有多Src的SP基因突变体的定向演进，以增强可溶性SP表达水平。为了容易地识别生长在培养皿上生长的菌落的可溶性SP表达水平，当其融合到SP基因突变体文库的30个末端时，使用MCHerry荧光蛋白作为Sp折叠的报告器。在三轮筛选后，与野生型和稀有密码子SP控制相比，含有九个Srcs的最佳SP基因突变体在可溶性蛋白表达水平上表现出大约六倍的增强。该研究表明，SRC的选择性引入可以在特定点衰减平均值，并且这种不连续衰减可以在时间上分离肽链的区段的平移，并主动协调其共转化折叠，导致功能性蛋白表达增强。 Biotechnol。 （c）2016 Wiley期刊，Inc。

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