title: Expression of cold-adapted β-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.
abstract: Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes.
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-Ku, Kobe, 657-8501, Japan.
authors.forename: Motonori
authors.initials: M
authors.lastname: Kudou
authors.forename: Fumiyoshi
authors.initials: F
authors.lastname: Okazaki
authors.forename: Nanami
authors.initials: N
authors.lastname: Asai-Nakashima
authors.forename: Chiaki
authors.initials: C
authors.lastname: Ogino
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001426:Bacterial Proteins / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D041965:Flavobacteriaceae / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D043325:Xylan Endo-1,3-beta-Xylosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C024278:protein S, Myxococcus xanthus; D001120:Arginine; D043325:Xylan Endo-1,3-beta-Xylosidase