Chloroplast Tag Modeling Comparison

Here we have modeled the locations of our engineered proteins without the signal peptide along with the naturally occurring form of the gene with the program SignalP. SignalP uses statistical analysis to predict N-terminal signal peptides and transit peptides. As shown, the wild type proteins that we used that are usually found in the thylakoid lumen are predicted to be targeted to the thylakoid lumen. In contrast, our engineered proteins are predicted to be targeted to ‘other’. This indicates that our engineered proteins lack a signal peptide and transit peptide, meaning they will be targeted to the cytosol.

In contrast, the two wild-type constructs that begin with CrtE are targeted to the cytosol. This is because wild-type CrtE does not have a signal peptide, meaning it is naturally targeted to the cytosol. This is because CrtE originates from Pantoea ananatis LMG 20103, which is a bacterium, so it does not contain chloroplasts or a thylakoid lumen. For the crtE-cytoTDS-MBP construct, the signal peptides of the cytoTDS are too far away from the N-Terminus to be recognized as a signal peptide.

We used SignalP for modeling, found here: https://services.healthtech.dtu.dk/service.php?TargetP

SignalP works by comparing input data to its library of both annotated and unannotated protein sequences across all life domains. Using their library of all five signal peptide types, SignalP is able to suggest where any encoded peptide will be transported to by cellular transport mechanisms. ¹

References

1. Teufel, F. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat. Biotechnol. 40, 1023–1025 (2022).