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

Yuji Kondo1,2, Yuxin Li1, Tetsuya Okajima1,2

  • 1Department of Molecular Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

Molecules (Basel, Switzerland)
|March 13, 2024
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel method to produce aggregated Notch1 EGF14-15 proteins, crucial for antibody development. This technique enhances the secretion and purification of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins.

Keywords:
Notch1Sparcl1secretion

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Glycobiology

Background:

  • Epidermal growth factor (EGF) repeats are structural motifs found in many proteins, including the Notch receptor.
  • These repeats contain unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc).
  • O-GlcNAc modification plays a role in various cellular processes, making its study important.

Purpose of the Study:

  • To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1.
  • To overcome challenges in producing and purifying the recombinant Notch1 EGF14-15 protein for use as an immunogen.
  • To establish a scalable method for obtaining O-GlcNAc modified Notch1 EGF14-15.

Main Methods:

  • Expression of recombinant C-terminal His6-tagged Notch1 EGF14-15 in HEK293T cells.
  • Fusion of Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15 to enhance secretion and prevent aggregation.
  • Utilizing PreScission protease for efficient release of Notch1 EGF14-15 from the fusion tag.
  • Generating deletion mutants of the escorting tag to optimize extracellular secretion.

Main Results:

  • Standard expression methods led to protein aggregation and poor secretion of Notch1 EGF14-15.
  • The Sparcl1 escorting tag facilitated efficient extracellular secretion of the fusion protein without aggregation.
  • Notch1 EGF14-15 was successfully purified and confirmed to be O-GlcNAcylated after cleavage from the tag.
  • Optimization of the escorting tag length improved the yield of secreted EGF14-15.

Conclusions:

  • The Sparcl1 escorting tag strategy is effective for producing soluble and purified O-GlcNAc modified Notch1 EGF14-15.
  • This method overcomes protein aggregation issues, enabling large-scale preparation of the target protein from cell culture supernatants.
  • The developed approach facilitates the generation of antibodies against specific glycan modifications on proteins like Notch1.