투고논문

Recombinant Production, Isotopic Labeling, and Preliminary NMR Characterization of a Hypothetical Protein from Galdieria sulphuraria

Young Kee Chae^1* and John L. Markley²

¹Department of Chemistry, Sejong University, Seoul, 05006, Republic of Korea

²Department of Biochemistry, University of Wisconsin – Madison, Madison, WI 53706, USA

Received May 2, 2025; Revised Jun 9, 2025; Accepted Jun 9, 2025

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 ¹H-¹⁵N 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 recombinant production, purification, isotopic labeling, HSQC, Galdieria sulphuraria

* Address correspondence to: Young Kee Chae, Department of Chemistry, Sejong University, Seoul, 05006, Republic of Korea, Tel: 82-6708-3748; Fax: 82-2-3408-4316; E-mail: ykchae@sejong.ac.kr