Purification of P0 myelin glycoprotein by a Cu2+-immobilized metal affinity chromatography.
P0 is an abundant myelin glycoprotein of peripheral nerves of vertebrates. Various point mutations of this protein are responsible for hereditary neuropathies. In this paper we described purification of P0 glycoprotein using SDS and a metal chelate affinity chromatography. Purified myelin fraction from bovine spinal roots in 0.5% SDS, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4 is filtered and applied directly to the Cu2+-immobilized affinity chromatography column, equilibrated with the same buffer. After eluting a void volume (or pass through) fraction, P0 protein was eluted by the same buffer but without salt. To remove contamination from the eluent, further purification is continued on a Concanavalin-A coupled agarose column. We purify within two days, 30 mg of P0 protein of apparent molecular weight 27 kDa. The method can be used to purify recombinant or mutated P0 protein found in severe pathologies.
Sedzik
J
J
Department of Physiology, Keio University School of Medicine, Tokyo, Japan.
Kotake
Y
Y
Uyemura
K
K
D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008970:Molecular Weight; D018993:Myelin P0 Protein / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D002241:Carbohydrates; D002614:Chelating Agents; D003902:Detergents; D008055:Lipids; D018993:Myelin P0 Protein; D012967:Sodium Dodecyl Sulfate; D006639:Histidine; D003300:Copper