Perriman R, Ares M
Center for Molecular Biology of RNA, Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
RNA. 1998 Sep;4(9):1047-54. doi: 10.1017/s135583829898061x.
Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, we have produced circular messenger RNAs with infinite open reading frames. We show that a circular mRNA containing a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular mRNA are fluorescent. Analysis of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA. This technology provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.
许多具有异常结构特性的蛋白质由多个重复的氨基酸序列组成，并且在重组系统中表达时往往不稳定。为了便于重组生产此类蛋白质用于结构和工程研究，我们制备了具有无限开放阅读框的环状信使核糖核酸（mRNA）。我们表明，含有简单绿色荧光蛋白（GFP）开放阅读框的环状mRNA能够在大肠杆菌中指导GFP的表达。具有无限GFP开放阅读框的环状mRNA产生极长的蛋白质链，证明细菌核糖体能够在环状mRNA内部起始并反复进行翻译。从环状mRNA产生的GFP只有单体形式具有荧光。对翻译起始区域的分析表明，多个序列有助于从环状mRNA实现最大程度的翻译。这项技术提供了一种生产非常长的重复序列蛋白质的独特方法，并可能为开发具有新特性的蛋白质材料开辟道路。
