# Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1

## TL;DR

Researchers developed a method to efficiently produce a specific part of the Notch protein that tends to aggregate, using a helper protein to improve secretion and purity.

## Contribution

A novel escorting strategy using Sparcl1 improves the extracellular secretion of aggregation-prone Notch EGF repeats.

## Key findings

- Fusing Sparcl1 to Notch1 EGF14-15 enabled efficient extracellular secretion without aggregation.
- Notch1 EGF14-15 was successfully released from the escorting tag and confirmed to be O-GlcNAcylated.
- Optimal escorting tag length was determined to enhance secretion of EGF14-15.

## Abstract

Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.