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首页> 外文期刊>Biotechnology Progress >Synthetic chimeras with orthogonal ribosomal proteins increase translation yields by recruiting mRNA for translation as measured by profiling active ribosomes
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Synthetic chimeras with orthogonal ribosomal proteins increase translation yields by recruiting mRNA for translation as measured by profiling active ribosomes

机译：具有正交核糖体蛋白的合成嵌合体可通过募集可翻译的mRNA（通过对活性核糖体进行谱分析来测量）来提高翻译产量

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In addition to their roles in protein biosynthesis, components of cellular ribosomes perform roles that contribute to a number of important cellular processes. Exploitation of processes has led to the use of ribosomal parts as solubility enhancer partners and purification matrices in protein expression. In this work, an engineered version of the E. coli ribosomal protein L29 (L4H2) as a fusion partner for enhancing cellular expression of proteins that are poorly expressed in bacteria was exploited. It was demonstrated that a chimeric fusion of L4H2 with various Fc receptors increases total expression up to 3.2-fold, relative to Fc receptors expressed without the fusion. Mechanistic insights using a novel application of in vivo ribosome display suggested that, although total cellular mRNA levels of L4H2-Fc receptor remained unchanged relative to wild-type Fc receptors, mRNA levels of actively translated L4H2-Fc transcript increased about 3.8-fold relative to actively translated levels of wild-type Fc transcript. Similar increases in protein expression in the context of the other proteins tested, showing the generality of this approach for proteins beyond human receptors was observed. These results extended the number of potential schemes by which orthogonal ribosomal parts can be used to enhance complex protein expression in bacterial platforms. Within a larger scope, this study features the possibility of engineering 5 tags that enhance mRNA affinity to ribosomes as strategies to augment translation. It was envisioned that the successful application of profiling active ribosomes in a highly targeted manner could be beneficial for mechanistic translation studies concerning synthesis of target proteins. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:285-293, 2016
机译：除了它们在蛋白质生物合成中的作用外，细胞核糖体的成分还发挥着有助于许多重要细胞过程的作用。工艺的开发已导致使用核糖体部分作为蛋白质表达中的溶解度增强伴侣和纯化基质。在这项工作中，利用了工程化形式的大肠杆菌核糖体蛋白L29（L4H2）作为融合伴侣，以增强细菌中表达差的蛋白的细胞表达。已经证明，与没有融合的Fc受体相比，L4H2与各种Fc受体的嵌合融合体将总表达增加了多达3.2倍。使用体内核糖体展示的新型应用的机制研究表明，尽管相对于野生型Fc受体，L4H2-Fc受体的总细胞mRNA水平保持不变，但相对于野生型Fc受体，活性翻译的L4H2-Fc转录物的mRNA水平相对于野生型Fc受体增加了约3.8倍主动翻译的野生型Fc转录水平。在测试的其他蛋白质的背景下，蛋白质表达也有类似的增加，这表明该方法对人类受体以外的蛋白质具有普遍性。这些结果扩展了潜在方案的数量，通过该方案，可以使用正交核糖体部分增强细菌平台中复杂蛋白的表达。在更大的范围内，这项研究的特征是工程化5种增强mRNA与核糖体亲和力的标签作为增加翻译的策略。可以预见，以高度针对性的方式成功分析活性核糖体可能对涉及目标蛋白合成的机械翻译研究有益。 （c）2016美国化学工程师学会生物技术学会。 Prog。，32：285-293，2016

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