Abstract
Background
Although hundreds of different signal peptides have now been identified, few studies have examined the factors enabling signal peptides to augment secretion of mature proteins. Signal peptides, located at the N‐terminus of nascent secreted proteins, characteristically have three domains: (1) a basic domain at the N‐terminus, (2) a central hydrophobic core, and (3) a carboxy‐terminal cleavage region. In this study, we investigated whether alterations in the basic and/or the hydrophobic domains of a commonly used signal peptide from interleukin‐2 (IL‐2) affected secretion of two proteins: placental alkaline phosphatase (AP) and endostatin.
Methods
A series of modifications in the basic and/or hydrophobic domains of the IL‐2 signal peptide were made by polymerase chain reaction with endostatin or AP plasmids as templates. Transfection of wild‐type or modified IL‐2 signal peptides fused in‐frame with endostatin or AP were done with Superfect in vitro or by the hydrodynamic method in vivo.
Results
Increasing both the basicity and hydrophobicity of the signal peptide augmented the secretion of AP and endostatin by approximately 2.5‐ and 3.5‐fold, respectively, from MDA‐MB‐435 cells in vitro. Over a range of DNA concentrations and times, the most effective IL‐2 signal peptide increased AP levels in the medium compared to the wild‐type IL‐2 signal peptide. Comparable results from these modified IL‐2 signal peptides were found to increase AP levels in the medium from bovine aortic endothelial cells. Moreover, the combined changes in basic and hydrophobic domains of the IL‐2 signal peptide augmented serum levels of endostatin and placental AP by 3‐fold when the optimal plasmid constructs were injected in vivo.
Conclusions
Modification of the IL‐2 signal peptide augments protein secretion both in vitro and in vivo. As a result, optimizing the signal peptide should be considered for increasing the therapeutic levels of secreted proteins. Copyright © 2004 John Wiley & Sons, Ltd.