title: Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification.
abstract: We report the engineering of a DnaE intein able to catalyze rapid C-terminal cleavage in the absence of N-terminal cleavage. A single mutation in DnaE intein from Nostoc punctiforme PCC73102 (NpuDnaE), Asp118Gly, was introduced based on sequence alignment with a previously engineered C-terminal cleaving intein mini-MtuRecA. This mutation was able to both suppress N-terminal cleavage and significantly elevate C-terminal cleavage efficiency. Molecular modeling suggests that in NpuDnaE Asp118 forms a hydrogen bond with the penultimate Asn, preventing its spontaneous cyclization prior to N-terminal cleavage. Mutation of Asp118 to Gly essentially abolishes this restriction leading to subsequent C-terminal cleavage in the absence of N-terminal cleavage. The Gly118 NpuDnaE mutant exhibits rapid thio-dependent C-terminal cleavage kinetics with 80% completion within 3 h at room temperature. We used this newly engineered intein to develop both column-free and chromatography-based protein purification methods utilizing the elastin-like-polypeptide and chitin-binding protein as removable purification tags, respectively. We demonstrate rapid target protein purification to electrophoretic purity at yields up to 84 mg per liter of Escherichia coli culture.
authors.affiliation: Artie McFerrin Department of Chemical Engineering,  Texas A&M University, College Station, TX 77843,  USA.
authors.forename: Miguel
authors.initials: M
authors.lastname: Ramirez
authors.forename: Najla
authors.initials: N
authors.lastname: Valdes
authors.forename: Dongli
authors.initials: D
authors.lastname: Guan
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011232:Chemical Precipitation; D002686:Chitin / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D004549:Elastin / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D047668:Inteins; D008958:Models, Molecular; D046937:Nostoc / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017354:Point Mutation; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D002686:Chitin; D004549:Elastin