title: Purification of green fluorescent protein using a two-intein system.
abstract: A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.
authors.affiliation: College of Biological Sciences, China Agricultural University, Beijing, 100094, China. gill2015@hotmail.com
authors.forename: Zhonglin
authors.initials: Z
authors.lastname: Zhao
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.forename: Baoqing
authors.initials: B
authors.lastname: Dun
authors.forename: Dan
authors.initials: D
authors.lastname: Jin
authors.forename: Shuzhen
authors.initials: S
authors.lastname: Ping
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.forename: Ming-Qun
authors.initials: MQ
authors.lastname: Xu
authors.forename: Min
authors.initials: M
authors.lastname: Lin
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D002942:Circular Dichroism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D047668:Inteins; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins