Comparing Oligomeric and Signal Motif ER Sequestration Strategies

Protein secretion is a powerful tool for therapeutic applications, enabling controlled delivery of drugs and other treatments. Temperature is an emerging input method that, unlike light or chemicals, can penetrate tissues and be applied with precision. To make this approach work, secreted proteins must be reliably retained in the endoplasmic reticulum (ER) to prevent unwanted “off-state” leakage. We designed and tested multiple protein constructs in HEK cells that used either signal motifs (KKXX and RXR, short sequences that normally keep proteins in the ER) or oligomerization domains (GCN4p1, HOTag3, and FTH1, which form clusters that block secretion). Secretion levels were measured in the absence of stimulation and normalized to protein expression. Among all strategies tested, the KKRN motif provided the strongest retention, producing the lowest background secretion while avoiding protein aggregation. These findings suggest that KKRN is a promising strategy for constructing reliable, temperature-controlled protein secretion systems.