Kondo, Y.; Li, Y.; Okajima, T. Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1. Molecules2024, 29, 1031.
Kondo, Y.; Li, Y.; Okajima, T. Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1. Molecules 2024, 29, 1031.
Kondo, Y.; Li, Y.; Okajima, T. Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1. Molecules2024, 29, 1031.
Kondo, Y.; Li, Y.; Okajima, T. Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1. Molecules 2024, 29, 1031.
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-His6. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, EGF14-15-His6 was efficiently released from the escorting tag. EGF14-15-His6 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-His6. Hence, a large amount of EGF14-15-His6 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.
Keywords
Notch1; Sparcl1; secretion
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
