title: Automated 96-well purification of hexahistidine-tagged recombinant proteins on MagneHis Ni(2)+-particles.
abstract: Functional genomics and the application of high-throughput (HT) approaches to solve biological and medical questions are the main drivers behind the increasing need for HT parallel expression and purification of recombinant proteins. Automation is necessary to facilitate this complex multistep process. We describe, in detail, an HT-automated purification of hexahistidine-tagged recombinant proteins using MagneHis Ni-Particles and the Biomek FX robot. This procedure is universally applicable to hexa-histidine-tagged recombinant proteins with the tag positioned at either the N- or C-terminus. With minor modifications, the automated protein purification protocol presented in this chapter could be adapted to purify recombinant proteins bearing other tags than hexahistidine and/or other expression systems than E. coli.
authors.affiliation: Molecular Biosciences, Pacific Northwest National Laboratory, Richland, WA, USA.
authors.forename: Chiann-Tso
authors.initials: CT
authors.lastname: Lin
authors.forename: Priscilla A
authors.initials: PA
authors.lastname: Moore
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kery
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D008280:Magnetics; D008863:Microspheres; D009842:Oligopeptides / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012371:Robotics
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine