The Department of Cell Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037, USA.
Mol Biotechnol. 2011 May;48(1):60-75. doi: 10.1007/s12033-010-9348-4.
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.
为了开发微藻作为生产有价值蛋白质的强大系统，我们分析了影响绿色藻类莱茵衣藻叶绿体中重组蛋白表达的一些因素。我们监测了三种密码子优化的转基因的 mRNA 积累、蛋白质合成和蛋白质周转，包括 GFP、细菌荧光素酶和一种大的单链抗体。GFP 和荧光素酶蛋白非常稳定，而抗体则不太稳定。相比之下，蛋白质合成的测量清楚地表明，当与相同的 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，这可以用有限的 mRNA 可用性来解释，因为在大多数情况下，与 atpA mRNA 相比，重组 mRNA 的积累相当好。体外足迹分析和体内多核糖体分析表明，核糖体结合减少可能导致翻译效率有限。然而，当整体分析重组多核糖体水平和蛋白质合成时，很明显，其他步骤，如蛋白质延伸效率低下，可能会产生相当大的影响。总之，我们的结果表明，在莱茵衣藻叶绿体中，翻译是限制异源蛋白表达的主要步骤。
