One-step purification of a functional, constitutively activated form of visual arrestin

• Published in: Protein expression and purification 2012-03, Vol.82 (1), p.55-60
• Main Authors: Huang, Li, Mao, Xiang, Abdulaev, Najmoutin G., Ngo, Tony, Liu, Wei, Ridge, Kevin D.
• Format: Article
• Language: English
• Subjects: Affinity fusion-tag; Animals; Arrestin; Arrestin - chemistry; Arrestin - genetics; Arrestin - isolation & purification; Arrestin - metabolism; Automated chromatography; Cattle; Cloning, Molecular; Desensitization; Escherichia coli - genetics; Gene Expression; Genetic Vectors; Protein purification; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Rhodopsin; Rhodopsin - metabolism; Subtilisin; Subtilisin - chemistry; Subtilisin - genetics; Subtilisin - isolation & purification; Vision
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2011.11.007

► Visual arrestins were expressed in bacterial expression system. ► Visual arrestins were purified by utilizing Profinity eXact™ fusion-tag system. ► Purified arrestin (R175E) binds to light activated non-phosphorylated rhodopsin. Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ∼48kDa soluble protein. While crystal structures for the inactive, ‘basal’ state of various arrestins are available, the conformation of ‘activated’ arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact™ fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho∗). The results show that both prodomain/Profinity eXact™ fusion-tagged wild-type and R175E arrestins can be expressed to levels approaching 2–3mg/l in Luria–Bertani media, and that the processed, tag-free mature forms can be purified to near homogeneity using a Bio-Scale™ Mini Profinity eXact™ cartridge on the Profinia™ purification system. Functional analysis of R175E arrestin generated using this approach shows that it binds to non-phosphorylated rhodopsin in a light-dependent manner. These findings should facilitate the structure determination of this ‘constitutively activated’ state of arrestin 1 as well as the monitoring of conformational changes upon interaction with Rho∗.