Impact of the N-terminal Fragment on the Solubility of eIF4E1 Solanum tuberosum

Obtaining soluble eukaryotic proteins using bacterial expression systems remains a significant challenge. Despite the availability of various techniques to optimize protein expression in E. coli, eukaryotic proteins are frequently expressed in an insoluble form when produced in prokaryotic cells.

Genes of interest were cloned into expression vectors, pET22b, and modified pET32a, using the restriction-ligation method. BL21(DE3), BL21(DE3) Star, BL21(DE3)pRARE, Tuner(DE3), Origami, SHuffle®T7, and C41(DE3) strains were used for analytical induction. Molecular modeling and molecular dynamics simulations were employed to design the experiments and to interpret the resulting data.

To prevent the accumulation of a truncated form of potato eIF4E in inclusion bodies, we tested various E. coli strains and repositioned affinity tags from the C-terminus to the N-terminus of the protein. Only the full-length eIF4E was found to be soluble in the prokaryotic expression system. Based on the eIF4E model and molecular dynamics simulations, we proposed a potential explanation for the impact of the N-terminal fragment on protein solubility.

The interaction between the N-terminal fragment and the dorsal surface of eIF4E may prevent protein aggregation. This shielding of hydrophobic regions appears to be a key factor in reducing aggregation, thereby facilitating the expression of eIF4E in a soluble form.