title: Comparison of affinity tags for protein purification.
abstract: Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.
authors.affiliation: Center for Gene Regulation, Department of Biochemistry and Molecular Biology, 108 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802-1014, USA.
authors.forename: Jordan J
authors.initials: JJ
authors.lastname: Lichty
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Malecki
authors.forename: Heather D
authors.initials: HD
authors.lastname: Agnew
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Michelson-Horowitz
authors.forename: Song
authors.initials: S
authors.lastname: Tan
mesh_terms: D000345:Affinity Labels; D000818:Animals; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004330:Drosophila; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000345:Affinity Labels; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013762:Tetrahydrofolate Dehydrogenase; D011494:Protein Kinases