Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.
Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.
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DOI：
10.1186/1472-6750-13-67
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发表时间：
2013-08-22
影响因子：
3.5
通讯作者：
Ryan MD
中科院分区：
文献类型：
--
作者：
Minskaia E;Nicholson J;Ryan MD
许多生物医学应用需要表达或生产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调共表达来完成。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 末端介导共翻译“切割”，因此编码多个蛋白质的序列（通过 2A 连接）可以连接成单个 ORF：单个转基因。然而，已经表明，在某些情况下，较短版本的 F2A 的切割效率可能会受到紧邻 F2A 上游的某些基因序列的 C 末端的抑制。本文描述了优化 F2A 共表达策略的进一步工作。我们在 GFP 和 CherryFP“报告”蛋白之间插入了不同长度的 F2A（相互或串联排列）。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的共表达和不同长度的 F2A 的切割效率。通过对 F2A 序列进行详细“微调”，确定了 F2A 序列的最佳和次优长度。根据我们的数据和模型（根据该模型，2A 活性是其与核糖体出口通道相互作用的产物），我们建议对上游蛋白 C 末端不“敏感”的 F2A 序列长度，可成功用于生物医学应用中两种蛋白的共表达。
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. 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. 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.
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DOI：
10.1073/pnas.0710650105
发表时间：
2008-03-25
影响因子：
11.1
作者：
Baranick, Brian T.;Lemp, Nathan A.;Logg, Christopher R.
通讯作者：
Logg, Christopher R.
影响因子：
5.1
作者：
Ryan, MD;Donnelly, M;Gani, D
通讯作者：
Gani, D
影响因子：
4.5
作者：
de Felipe, P;Ryan, MD
通讯作者：
Ryan, MD
影响因子：
11.4
作者：
RYAN, MD;DREW, J
通讯作者：
DREW, J
影响因子：
--
作者：
Pfuetzner, Wolfgang
通讯作者：
Pfuetzner, Wolfgang