title: One-step purification of a functional, constitutively activated form of visual arrestin.
abstract: Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa 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 2-3 mg/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∗.
authors: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu Province 210095, China.
authors: Li
authors: L
authors: Huang
authors: Xiang
authors: X
authors: Mao
authors: Najmoutin G
authors: NG
authors: Abdulaev
authors: Tony
authors: T
authors: Ngo
authors: Wei
authors: W
authors: Liu
authors: Kevin D
authors: KD
authors: Ridge
mesh_terms: D000818:Animals; D019393:Arrestin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D019393:Arrestin; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin; D020860:Subtilisin