title: Complete removal and exchange of sodium dodecyl sulfate bound to soluble and membrane proteins and restoration of their activities, using ceramic hydroxyapatite chromatography.
abstract: Up to now, removal of sodium dodecyl sulfate (SDS) from proteins in terms of restoration of their activity was an unsolved problem. A general procedure using ceramic hydroxyapatite (HAP) chromatography was developed for the complete removal of SDS bound to soluble or membrane proteins. This procedure involves (i) the binding of the SDS-protein complexes onto the ceramic hydroxyapatite column, (ii) extensive washing of bound proteins with phosphate buffer containing a mild detergent to exchange SDS, (iii) elution of the retained protein by increasing the phosphate concentration. Using this approach, complete exchange of [35S]SDS into a nonionic detergent such as dodecyl maltoside was achieved with a 90-100% protein recovery. The efficiency of protein-bound SDS removal is very likely due to the combined effect of phosphate ions and the hydrophobic tail of nonionic detergent: acting together, they are able to displace SDS molecules from their protein-binding sites. The advantages of this HAP-mediated SDS removal method include high efficiency, rapidity, simplicity and general applicability to a wide variety of detergents and soluble or membrane proteins. Of utmost importance, SDS-treated P-glycoprotein, glutamate dehydrogenase, and lysozyme fully recovered their enzymatic activities after HAP chromatography, including lysozyme electroeluted from SDS-polyacrylamide gel electrophoresis. This demonstrates that reactivation of SDS-treated protein can be achieved, provided that SDS is completely removed under mild conditions.
authors.affiliation: Institut de Biologie et Chimie des Protéines, UPR 412 CNRS, Lyon, France.
authors.forename: M
authors.initials: M
authors.lastname: Dong
authors.forename: L G
authors.initials: LG
authors.lastname: Baggetto
authors.forename: P
authors.initials: P
authors.lastname: Falson
authors.forename: M
authors.initials: M
authors.lastname: Le Maire
authors.forename: F
authors.initials: F
authors.lastname: Penin
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002516:Ceramics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003902:Detergents / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification; D006882:Hydroxyapatites; D008565:Membrane Proteins / Q000302:isolation & purification*; D009113:Muramidase / Q000302:isolation & purification; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003902:Detergents; D006882:Hydroxyapatites; D008565:Membrane Proteins; D011506:Proteins; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D005969:Glutamate Dehydrogenase; D009113:Muramidase