Improvement
Through the results of previous experiments, as well as ancillary analysis of protein structure predictions, we concluded that the T2A peptides we used affects protein structure regardless of whether it is broken or not. This is because through modeling we found that the junctions between the T2A peptides in the target protein and the main protein structure typically have a large fold angle, and this causes the T2A peptides to fold inward, rather than extending to the outside of the protein. As a result of the inward fold of the T2A peptides, in most cases, the structure of our target protein changes, which affects the activity of the protein. This is something we do not want to see.
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In order to be able to use 2A peptides without affecting the function of the target protein, we wanted to find 2A peptides that would not significantly alter the protein structure to replace the T2A peptides we currently use. There is a literature[1] has investigated the breaking efficiency of different 2A peptides, from which we selected 10 groups of 2A peptides (ERBV-1, PTV, OpbuCPV18, PnPV2A1, EMCV, BmCPV1, PrV2A1, ERAV, PnPV2A2, FMDV) with the highest break efficiency as our alternatives. Using a structure prediction approach, we initially determined whether these 10 groups of 2A peptides we selected have a negative impact on the structure of the target protein.
Fig 2. Analysis of 2A peptide ‘cleavage’ efficiency.[1] (a) ‘Cleavage’ efficiency, as a percentage for the 22 different 2A peptides tested. Data shown represent the intensity average values ± standard deviation of western blotting of three independent biological replicates. (b) Alignment among 2A peptides used in this work showing conserved residues. There is high conservation in the motif DXEXNPGP important for ‘cleavage’ efficiency (shown as dark shaded).
By respectively combining yeGFP and 10 groups of 2A peptides (yeGFP-ERAV, yeGFP-BmCPV1, yeGFP-PTV, yeGFP-PnPV2A2, yeGFP-PnPV2A1, yeGFP-FMDV, yeGFP-OpbuCPV1, yeGFP-EMCV, yeGFP- ERBV-1 and yeGFP-PrV2A1), we obtained 10 yeGFP-2A structures. By using the I-TASSER server to predict the structure of these proteins, we obtained the following results.
Fig 3. I-TASSER prediction structure. (a) yeGFP-ERAV. (b) yeGFP-BmCPV1. (c) yeGFP-PTV. (d) yeGFP-PnPV2A2. (e) yeGFP-PnPV2A1. (f) yeGFP-FMDV. (g) yeGFP-OpbuCPV1. (h) (i) yeGFP-EMCV. (i) yeGFP-ERBV-1. (j) yeGFP-PrV2A1. The red part is the 2A peptide portion. The white part is the spacer peptides (GWEGRA) linking yeGFP and 2A peptide.
The predicted results showed that among the 10 groups of 2A peptides we selected with high breakage efficiency, five 2A peptides (ERAV, BmCPV1, PTV, PnPV2A2, and PnPV2A1) were not folded inward toward the inner side of the protein structure but extended toward the outer side of the protein structure. This indicates that these may not significantly alter the structure of the target proteins and are expected to replace the T2A peptides we used previously. To test this conclusion, we could experimentally test the actual effect of 2A peptides on the activity of our target proteins.