Enhanced large-scale production and purification of recombinant human NUPR1 for detailed structural and functional...

Nuclear Protein 1 (NUPR1) is a critical modulator of numerous cellular processes, including cell cycle regulation, apoptosis, and oncogenic transformation. Its structural characterization is indispensable for understanding its multifaceted biological functions and for its validation as a potential therapeutic target in diseases. Although foundational studies have reported on the expression and purification of NUPR1, detailed and scalable protocols for large-scale production, a prerequisite for advanced biophysical techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy, have remained largely unarticulated. This study describes a successfully developed, optimized, and scalable protocol for the high-yield production and purification of recombinant human NUPR1. This optimized protocol consistently yielded 2-3 mg of NUPR1 per liter of M9 minimal medium with greater than 95% purity and high monodispersity, as confirmed by SEC-MALS analysis, making it highly suitable for demanding structural biology applications. Furthermore, efficient isotopic labeling strategies incorporating ¹⁵N and ¹³C were successfully implemented, enabling detailed multi-dimensional NMR spectroscopic analysis. Preliminary NMR data, including backbone resonance assignments, unequivocally confirmed the intrinsically disordered nature of NUPR1. Secondary structure propensity analysis derived from these assignments indicated a characteristically low propensity for the formation of stable canonical secondary structures. This robust, well-characterized, and scalable expression and purification pipeline provides an essential and solid foundation for future detailed structural, dynamic, and functional investigations of NUPR1, which are crucial for dissecting its complex roles in cellular physiology and disease pathogenesis.