Complete removal and exchange of sodium dodecyl sulfate bound to soluble and membrane proteins and restoration of their activities, using ceramic hydroxyapatite chromatography.
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.
Dong
M
M
Institut de Biologie et Chimie des Protéines, UPR 412 CNRS, Lyon, France.
Baggetto
L G
LG
Falson
P
P
Le Maire
M
M
Penin
F
F
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
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