Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast.
Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast.
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分子因素影响重组蛋白在衣原体中的重组蛋白的积累。
DOI：
10.1007/s12033-010-9348-4
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发表时间：
2011-05
影响因子：
2.6
通讯作者：
Mayfield, Stephen P.
中科院分区：
文献类型：
--
作者：
Coragliotti, Anna T.;Beligni, Maria Veronica;Franklin, Scott E.;Mayfield, Stephen P.
关键词：
在努力发展微藻作为一个强大的系统，生产有价值的蛋白质，我们分析了一些影响重组蛋白表达的绿色衣藻的叶绿体中的因素。我们监测了三种密码子优化的转基因（包括GFP、细菌荧光素酶和大单链抗体）的mRNA积累、蛋白质合成和蛋白质周转。GFP和荧光素酶蛋白相当稳定，而抗体则不太稳定。相反，蛋白质合成的测量清楚地表明，当与相同atpA启动子/UTR控制下的内源性mRNA相比时，三种嵌合mRNA的翻译大大减少。只有在少数情况下，这可以解释为有限的mRNA的可用性，因为在大多数情况下，重组mRNA积累相当不错，相比atpA mRNA。在体外toeprint和体内多核糖体分析表明，减少核糖体协会可能有助于有限的翻译效率。然而，当重组多核糖体水平和蛋白质合成作为一个整体进行分析时，很明显，其他步骤，如低效的蛋白质延伸，可能会产生相当大的影响。总之，我们的结果表明翻译是限制异源蛋白在C.莱氏叶绿体
In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.
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影响因子：
5.5
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通讯作者：
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影响因子：
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