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

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.