title: Contamination from an affinity column: an encounter with a new villain in the world of membrane-protein crystallization.
abstract: Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin. The contamination of protein solutions purified on Strep-Tactin columns has never been described previously and seems to be specific to membrane proteins solubilized in detergents. Trace amounts of Strep-Tactin were observed to be eluted from a Strep-Tactin column using several routinely used detergents, illustrating their possible role in the contamination. This finding raises an alarm and suggests caution in membrane-protein purification using Strep-Tactin affinity columns, where detergents are essential components. The small crystals of contaminant protein led to the structure at 1.9 Å resolution of Strep-Tactin in complex with desthiobiotin.
authors.affiliation: CEA, Institut de Biologie Structurale Jean-Pierre Ebel, 41 Rue Jules Horowitz, 38027 Grenoble, France.
authors.forename: Pankaj
authors.initials: P
authors.lastname: Panwar
authors.forename: Aurélien
authors.initials: A
authors.lastname: Deniaud
authors.forename: Eva
authors.initials: E
authors.lastname: Pebay-Peyroula
mesh_terms: D029681:Arabidopsis Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003460:Crystallization; D018360:Crystallography, X-Ray; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D019809:Streptavidin / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D001710:Biotin; C004749:desthiobiotin; D019809:Streptavidin