Xentry 9 2019 is a powerful diagnostic software for Mercedes-Benz vehicles. However, this article focuses on a different “Xentry,” a cell-penetrating peptide (CPP) used in a scientific study to optimize the production of chimeric Green Fluorescent Protein (GFP) and CPP fusion proteins. This research explored various CPPs and modifications to enhance cellular uptake and delivery of proteins into living cells.
Researchers initially used R10, a CPP consisting of ten arginine residues, fused to superfolder GFP (sfGFP). However, expression in E. coli TOP10 cells failed to produce the desired R10-sfGFP. Switching the tag positions to sfGFP-R10 resulted in truncated proteins, likely due to protease activity within the cells. Similar truncations were observed with other CPPs like TAT and Transportan.
To mitigate proteolytic cleavage, the study shifted to E. coli BL21(DE3)pLysS and the pEV vector system. This system successfully produced full-length eGFP-R8, leading to the adoption of R8 and eGFP as the model CPP and cargo, respectively. Subsequently, various CPPs (Penetratin, Transportan, Xentry, TAT) were successfully fused to eGFP, yielding full-length proteins. However, Integrin and MAP constructs resulted in truncated proteins. Interestingly, eGFP-Xentry predominantly formed dimers due to disulfide bond formation.
Figure 1: SDS-PAGE analysis of purified eGFP-CPP fusion proteins.
Cellular uptake of the eGFP-CPP fusion proteins was assessed using confocal microscopy and flow cytometry. HeLa cells treated with eGFP lacking a CPP showed no internalization, confirming the CPP’s role in cellular entry. eGFP-R8 and eGFP-Transportan exhibited significant uptake, albeit with distinct subcellular distributions. eGFP-R8 accumulated in the perinuclear region, while eGFP-Transportan localized in punctate structures at the cell periphery. eGFP-Penetratin and eGFP-TAT also showed perinuclear enrichment but with lower uptake levels. The punctate patterns suggested endocytosis as the primary uptake mechanism.
Flow cytometry analysis across various cell lines (HeLa, HEK, 10T1/2, HepG2) revealed cell-type and CPP-dependent uptake variations. 10T1/2 cells displayed the highest uptake for cationic peptides (Penetratin, R8, TAT). Notably, eGFP-Transportan showed exceptionally high uptake in HepG2 cells. Overall, Transportan consistently outperformed other CPPs in terms of cellular internalization.
Figure 2: Confocal microscopy images of HeLa cells treated with eGFP-CPP fusion proteins.
To further enhance cellular uptake, researchers investigated CPP modifications, including cyclization and the addition of an endosomal escape sequence (HA) from the influenza virus. Cyclization involved introducing cysteine residues flanking the CPP sequence to form a disulfide bond. eGFP-HA-TAT and cyclic versions of Penetratin, R8, and TAT (cPenetratin, cR8, cTAT) were successfully produced. However, eGFP-cTransportan proved challenging to express and purify.
Confocal microscopy and flow cytometry demonstrated that cyclization or HA addition significantly increased cellular uptake. Cyclization altered the subcellular localization of eGFP-R8 and eGFP-TAT, shifting from perinuclear to a more peripheral distribution, resembling eGFP-Transportan. eGFP-cTAT showed a dispersed cellular distribution, while eGFP-HA-TAT exhibited some perinuclear enrichment. Cyclization of R8 and TAT consistently enhanced uptake across all cell lines, with HeLa cells showing the most significant increase. While HA addition to TAT also improved uptake, it was less effective than cyclization. Even at higher concentrations (100 µM), cyclic CPPs primarily entered cells via endocytosis.
In conclusion, this study highlights the successful recombinant production of chimeric GFP and CPP fusion proteins, particularly using the Xentry CPP. Furthermore, it demonstrates that CPP modifications like cyclization and HA addition can significantly improve cellular uptake, paving the way for more efficient protein delivery into cells.
