Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues

Abstract

Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A “cleavage” may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites) used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.

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

“…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. 38 The different cleavage efficiencies observed for these 2A peptides could be due to the changes in the experimental designs.…”

“…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. 38 The different cleavage efficiencies observed for these 2A peptides could be due to the changes in the experimental designs.…”

Construction of recombinant adenovirus containing picorna-viral 2A-peptide sequence for the co-expression of neuro-protective growth factors in human umbilical cord blood cells

“…The variations in cleavage activity of the various lengths of 2A-peptides are attributed to the nature of the region immediately upstream of CHYSEL (18-30 amino acid upstream of the cleavage site). 21 Using combination of furin recognition site and 2A-peptide strategy of expression has shown stable co-expression of transgene in different cell lines. 22 Although the 2A system is appropriate for delivering co-expression of particular sets of transgenes, it is not suited in situations where individual transgenes require a different tissue distribution.…”

Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality

“…2a, see lane 2 at various time points). As the efficiency of 2A skipping is known to be dependent on the amino acid context (Minskaia & Ryan, 2013), it appears that the amino acid sequence adjacent to the P2A peptide in our recombinant NiV is not conducive to efficient skipping. It is known that addition of a GSG linker immediately upstream of the skipping sequence improves skipping efficiency (Holst et al, 2006;Szymczak et al, 2004), presumably by allowing the 2A peptide to favour the conformation that induces ribosomal skipping.…”