Enhancing functional expression of codon‐optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons
Enhancing functional expression of codon‐optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons
复制标题
DOI：
10.1002/bit.26238
复制
发表时间：
2017-05
影响因子：
3.8
通讯作者：
Chao Zhong;P. Wei;Y. Zhang
中科院分区：
文献类型：
--
作者：
Chao Zhong;P. Wei;Y. Zhang
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、氨基酸的错误掺入和/或mRNA的缓慢翻译，从而降低异源蛋白质的表达。然而，它的假设功能有关的功能性蛋白质折叠几乎没有报道。在这里，我们研究了选择性引入同义稀有密码子（SRC）对两个密码子优化（即，稀有无密码子）基因蔗糖磷酸化酶（SP）基因和酰胺水解酶基因在大肠杆菌BL 21（DE 3）中的表达水平。我们研究了在罕见的无密码子sp和ah基因的前半部分的α螺旋、β链或接头的编码区中引入单个SRC。与其他区域相比，在β链编码区的起始处引入单个SRC大大增强了其可溶性表达水平。此外，我们应用定向进化来测试含有多个SRC的sp基因突变体以增强可溶性SP表达水平。为了容易地鉴定培养皿上生长的菌落的可溶性SP表达水平，当mCherry荧光蛋白融合到sp基因突变体文库的3′端时，将其用作SP-折叠报告基因。经过三轮筛选，与野生型和罕见的无密码子sp对照相比，含有9个SRC的最佳sp基因突变体在可溶性蛋白表达水平上表现出约6倍的增强。这项研究表明，选择性引入SRC可以减弱特定点的翻译，这种不连续的减弱可以暂时分离肽链片段的翻译，并积极协调它们的共翻译折叠，从而增强功能性蛋白质的表达。Biotechnol. Bioeng. 2017;114：1054-1064.© 2016 Wiley Periodicals，Inc.
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 3′ 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. Bioeng. 2017;114: 1054–1064. © 2016 Wiley Periodicals, Inc.