Automated 96-well purification of hexahistidine-tagged recombinant proteins on MagneHis Ni(2)+-particles.
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.
Lin
Chiann-Tso
CT
Molecular Biosciences, Pacific Northwest National Laboratory, Richland, WA, USA.
Moore
Priscilla A
PA
Kery
Vladimir
V
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
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine