Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicagosativa.

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/μg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/μg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.