Sacchetti A, El Sewedy T, Nasr A F, Alberti S
Laboratory of Experimental Oncology and Biotech Group, Department of Cell Biology and Oncology, Instituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy.
FEBS Lett. 2001 Mar 9;492(1-2):151-5. doi: 10.1016/s0014-5793(01)02246-3.
Green fluorescent protein (GFP) variants with higher expression efficiencies have been generated by mutagenesis. Favorable mutations often improve the folding of GFP. However, an effect on protein folding fails to explain the efficiency of several other GFP mutations. In this work, we demonstrate that mutations of the GFP open reading frame and untranslated regions profoundly affect mRNA transcription and translation efficiencies. The removal of the GFP 5' untranslated region halves the transcription rate of the GFP gene, but hugely improves its translation rate. Mutations of the GFP open reading frame or the addition of peptide sequences differentially reduce the GFP mRNA transcription rate, translation efficiency and protein stability. These previously unrecognized effects are demonstrated to be critical to the efficiency of GFP mutants. These findings indicate the feasibility of generating more efficient GFP variants, with optimized mRNA transcription and translation in eukaryotic cells.
通过诱变已经产生了具有更高表达效率的绿色荧光蛋白（GFP）变体。有利的突变通常会改善GFP的折叠。然而，对蛋白质折叠的影响并不能解释其他几种GFP突变的效率。在这项工作中，我们证明GFP开放阅读框和非翻译区的突变会深刻影响mRNA转录和翻译效率。去除GFP 5'非翻译区会使GFP基因的转录速率减半，但会极大地提高其翻译速率。GFP开放阅读框的突变或肽序列的添加会不同程度地降低GFP mRNA转录速率、翻译效率和蛋白质稳定性。这些先前未被认识到的影响被证明对GFP突变体的效率至关重要。这些发现表明在真核细胞中通过优化mRNA转录和翻译来产生更高效的GFP变体是可行的。
