Use of tandem‐affinity chromatography to incorporate nucleotide loading of small <scp>GTPases</scp> during their purification

2026/05/17 by Rofiat Oladimeji, Chigozie Odo, Ethan Lewis +3
Biochemistry, Genetics and Molecular Biology · Materials Science · #Enzyme Structure and Function #Protein Structure and Dynamics #RNA and protein synthesis mechanisms

paper · doi:10.1002/pro.70628

openalex publication_date 2026/05/17 · openalex created_date 2026/05/19 · openalex updated_date 2026/07/30

Abstract

A bottleneck for structure-function studies of closely related proteins is purification. Here, we built upon new affinity protein purification technology to develop a uniform and rapid tandem-affinity purification protocol to generate significant quantities of pure GTPase proteins for use in structure-function studies. Our method relies on new and commercially available split-intein based technology. We show that this technology can be used to speed up purification of small GTPases and at the same time facilitate nucleotide loading. Using K-Ras4B and H-Ras as case studies, we demonstrate that we can generate crystallizable grade protein, homogenously loaded with either GDP or the non-hydrolysable GTP analogue GppNHp, in less than 36 h.

Citations

• Structure-Function Relationships of the G Domain, a Canonical Switch Motif
• Accurate structure prediction of biomolecular interactions with AlphaFold 3

Related

• Simplifying recombinant protein production: Combining Golden Gate cloning with a standardized protein purification scheme
• Need for speed: advances in the era of high throughput interaction proteomics
• Host cell protein networks as a novel co‐elution mechanism during protein A chromatography
• A nanobody-based strategy for rapid and scalable purification of native human protein complexes
• Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry