Journal of the Korean Magnetic Resonance Society (한국자기공명학회논문지)

Korean Magnetic Resonance Society (한국자기공명학회)
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Recombinant Production, Isotopic Labeling, and Preliminary NMR Characterization of a Hypothetical Protein from Galdieria sulphuraria

  • Young Kee Chae (Department of Chemistry, Sejong University) ;
  • John L. Markley (Department of Biochemistry, University of Wisconsin - Madison)
  • Received : 2025.05.02
  • Accepted : 2025.06.09
  • Published : 2025.06.23
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Abstract

Galdieria sulphuraria is a thermoacidophilic red alga that thrives in extreme environments and encodes numerous hypothetical proteins with uncharacterized functions. In this study, we describe the recombinant production, purification, and isotopic labeling of a truncated construct (residues 1-130) of the hypothetical protein Gasu_38600 for NMR-based structural analysis. The construct was expressed in Escherichia coli using a maltose-binding protein fusion system and purified via immobilized metal affinity chromatography followed by TEV protease cleavage. Uniform 15N and [13C,15N] isotopic labeling was achieved using minimal media supplemented with 15NH4Cl and 13C-glucose. High-quality 1H15N HSQC spectra indicated that the protein adopts a folded conformation in solution, supporting its suitability for further structural investigation. These results lay the groundwork for future studies to elucidate the function of this conserved hypothetical protein from an extremophilic organism.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea (NRF-2021R1F1A1046500). The author thanks Dr. Marco Tonelli of the NMRFAM for this technical assistance. This study made use of the National Magnetic Resonance Facility at Madison, an NIH Biomedical Technology Research Resource Center NIH R24GM141526. Helium recovery equipment, computers, and infrastructure for data archive were funded by the University of Wisconsin-Madison, NIH R24GM141526.

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