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

Abstract

BackgroundMany 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.ResultsWe 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.ConclusionsOptimal 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.

Adoption of the 2A Ribosomal Skip Principle to Tobacco Mosaic Virus for Peptide Display

“…The major disadvantage of the utilization of the FMDV 2A ribosomal skip sequence is the unpredictable ratio of fusion protein to CP (Luke et al, 2010; Minskaia et al, 2013), but this can be adjusted through the use of length-optimized variants of the 2A sequence (Minskaia et al, 2013). When displaying epitopes on the surface of a virus for immunization, it is better to choose a 2A sequence variant that achieves the optimal epitope-to-CP ratio thus maximizing the number of epitopes presented to the immune system.…”

Co-expression of functional human Heme Oxygenase 1, Ecto-5′-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by “self-cleaving” 2A peptide system

“…Whereas the addition of both furin recognition site and spacer upstream of F2A sequences in our system resulted in an inefficient production of our transgenes. To improve the 2A efficiency, recent efforts are aimed to identify the optimal length of the F2A sequence itself for successful co-expression strategies (Minskaia et al, 2013).…”

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

“…34 When evaluated in human cell lines, zebrafish and mice, P2A exhibited the highest cleavage efficiency followed by T2A, E2A and F2A. 32 The cleavage efficiency of a 2A peptide may be affected by the nature of the protein expressed, 33,35 the order of genes flanking 2A, 18,36 the length of the 2A peptide used, 33,37 and the linker between the upstream protein and 2A. 34,38,39 Inserting GSG and SGS linkers, V5 epitope tag (GKPUPNPLLGLDST) and 3xFlag epitope tag immediately preceding 2A has been shown to improve 2A cleavage efficiency, 34,39,40 while inserting an ad-hoc sequence for cloning purposes resulted in decreased cleavage efficiency.…”