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Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications

机译：用于生物医学应用的口蹄疫病毒2A共表达系统的优化

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Background Many biomedical applications require the expression or production of therapeutic hetero-multimeric proteins/protein complexes: in most cases only accomplished by co-ordinated co-expression within the same cell. Foot-and-mouth disease virus 2A (F2A) and ‘2A-like’ sequences are now widely used for this purpose. Since 2A mediates a co-translational ‘cleavage’ at its own C-terminus, sequences encoding multiple proteins (linked via 2As) can be concatenated into a single ORF: a single transgene. It has been shown that in some cases, however, the cleavage efficiency of shorter versions of F2A may be inhibited by the C-terminus of certain gene sequences immediately upstream of F2A. This paper describes further work to optimise F2A for co-expression strategies. Results We have inserted F2A of various lengths in between GFP and CherryFP ‘reporter’ proteins (in reciprocal or tandem arrangements). The co-expression of these proteins and cleavage efficiencies of F2As of various lengths were studied by in vitro coupled transcription and translation in rabbit reticulocyte lysates, western blotting of HeLa cell lysates and fluorescence microscopy. Conclusions Optimal and suboptimal lengths of F2A sequences were identified as a result of detailed ‘fine-tuning’ of the F2A sequence. Based on our data and the model according to which 2A activity is a product of its interaction with the exit tunnel of the ribosome, we suggest the length of the F2A sequence which is not ‘sensitive’ to the C-terminus of the upstream protein that can be successfully used for co-expression of two proteins for biomedical applications.
机译：背景技术许多生物医学应用都需要表达或产生治疗性异源多聚体蛋白质/蛋白质复合物：在大多数情况下，只能通过在同一细胞内协同表达来实现。口蹄疫病毒2A（F2A）和“ 2A样”序列现已广泛用于此目的。由于2A在其自身的C端介导共翻译的“切割”，因此可以将编码多个蛋白质（通过2A连接）的序列连接成单个ORF：单个转基因。然而，已经表明，在某些情况下，较短版本的F2A的切割效率可能会被F2A上游某些基因序列的C末端所抑制。本文介绍了针对共表达策略优化F2A的进一步工作。结果我们在GFP和CherryFP“报告基因”蛋白之间（互惠或串联排列）插入了各种长度的F2A。通过兔网织红细胞裂解物中的体外偶联转录和翻译，HeLa细胞裂解物的蛋白质印迹和荧光显微镜研究了这些蛋白质的共表达和各种长度的F2A的切割效率。结论通过对F2A序列进行详细的“微调”，可以确定F2A序列的最佳和次佳长度。根据我们的数据和模型，其中2A活性是其与核糖体出口隧道相互作用的产物，我们建议F2A序列的长度对上游蛋白的C端不敏感。可以成功用于生物医学应用中两种蛋白的共表达。

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