Protein expression and purification ·11 Nov 2026 ·Added 12 Nov 2026

TwinStrep™ tag purification leads to differences in RAF1 RAS binding domain and cysteine-rich domain conformation.

Gabriel Cornilescu, Shelley Perkins, Vanessa E Wall, Robert A D'Ippolito, Troy Taylor, Matthew Drew, Peter H Frank, Min Hong, Kelly Snead, Monalisa Swain, Caroline J Dehart, Andrew G Stephen, William Gillette, Dominic Esposito, Simon A Messing

DOI ↗

Abstract

Immobilized Metal Affinity Chromatography (IMAC) is widely used for recombinant protein purification. However, its reliance on metal ions raises concerns for metal-binding proteins, which constitute a significant fraction of all proteins. To analyze the effects of IMAC purification on metal-binding proteins, we designed a vector that replaces the His-tag with a streptavidin-based affinity tag, TwinStrep™. This new E. coli vector is used to produce TwinStrep™-MBP (maltose-binding protein)-TEV site (tobacco-etch-virus)-POI (protein of interest). Using this system, we purified KRAS4b G-domain, PI3Kα RAS-binding domain (RBD), and RAF1 RBD plus its cysteine-rich domain (CRD), and compared them to IMAC-purified counterparts through thermal melting, NMR, mass spectrometry, and biochemical assays. KRAS4b and RAF1 proteins are metal-binding proteins, while PI3Kα is not. While KRAS4b and PI3Kα showed no differences between purification methods, RAF1 exhibited distinct conformational changes when purified without metal exposure. These findings demonstrate that RAF1 clone design and purification strategy influences its structural properties, with possible implications for functional studies and therapeutic development.