A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.
We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.
Department of Chemical Engineering, University of Washington, Box 351750, Seattle, Washington, 98195-1750.
Brandon L
BL
Coyle
François
F
Baneyx
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012822:Silicon Dioxide / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D058619:Maltose-Binding Proteins; D010447:Peptide Hydrolases; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012822:Silicon Dioxide; D000097573:Red Fluorescent Protein; C499588:ompT protein, E coli
affinity tag; fusion protein; proteolytic cleavage; purification; silica