title: Use of streamline chelating for capture and purification of poly-His-tagged recombinant proteins.
abstract: Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6 x His-tagged protein binds to a chelating resin charged with metal ions such as Ni2+, Cu2+ or Zn2+, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S. cerevisiae and a modified diphtheria toxin produced intracellularly by E. coli, were expressed with 6 x His tags and could therefore be purified using IMAC. In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced. It was possible to capture the intracellular diphtheria protein from E. coli directly after cell lysis, without prior centrifugation or filtration. The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture. Detailed information on the experimental work performed, and the capture processes developed, is provided.
authors.affiliation: Biotechnology Unit, NIDDK, NIH, Bethesda, MD 20892, USA.
authors.forename: S
authors.initials: S
authors.lastname: Noronha
authors.forename: J
authors.initials: J
authors.lastname: Kaufman
authors.forename: J
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D000327:Adsorption; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004167:Diphtheria Toxin / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D017780:Malaria Vaccines / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D002614:Chelating Agents; D004167:Diphtheria Toxin; D017780:Malaria Vaccines; D011994:Recombinant Proteins; D006639:Histidine