title: Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent.
abstract: Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components. We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E. coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products. In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins. Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed. Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents.
authors.affiliation: Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands.
authors.forename: K L
authors.initials: KL
authors.lastname: Franken
authors.forename: H S
authors.initials: HS
authors.lastname: Hiemstra
authors.forename: K E
authors.initials: KE
authors.lastname: van Meijgaarden
authors.forename: Y
authors.initials: Y
authors.lastname: Subronto
authors.forename: J
authors.initials: J
authors.lastname: den Hartigh
authors.forename: T H
authors.initials: TH
authors.lastname: Ottenhoff
authors.forename: J W
authors.initials: JW
authors.lastname: Drijfhout
mesh_terms: D019840:2-Propanol; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D003902:Detergents / Q000302:isolation & purification; D004340:Drug Contamination; D004731:Endotoxins / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005293:Ferritins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017830:Octoxynol; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D050725:Papillomavirus E7 Proteins; D011092:Polyethylene Glycols / Q000302:isolation & purification; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012997:Solvents
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D004731:Endotoxins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D012997:Solvents; C059731:oncogene protein E7, Human papillomavirus type 16; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D011092:Polyethylene Glycols; D006639:Histidine; D017830:Octoxynol; D005293:Ferritins; C010615:Nonidet P-40; D019840:2-Propanol