Sedzik J, Kotake Y, Uyemura K
Department of Physiology, Keio University School of Medicine, Tokyo, Japan.
Neurochem Res. 1999 Jun;24(6):723-32. doi: 10.1023/a:1020723328143.
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.
P0是脊椎动物外周神经中一种丰富的髓磷脂糖蛋白。该蛋白的各种点突变可导致遗传性神经病。在本文中，我们描述了使用SDS和金属螯合亲和层析法纯化P0糖蛋白的过程。将来自牛脊髓根的纯化髓磷脂部分置于0.5% SDS、0.5 M NaCl、50 mM Tris-HCl（pH 7.4）中过滤，然后直接应用于用相同缓冲液平衡的固定化Cu2+亲和层析柱。洗脱空体积（或穿透）部分后，用相同但无盐的缓冲液洗脱P0蛋白。为了去除洗脱液中的污染物，继续在伴刀豆球蛋白A偶联琼脂糖柱上进一步纯化。我们在两天内纯化出了30 mg表观分子量为27 kDa的P0蛋白。该方法可用于纯化在严重病症中发现的重组或突变P0蛋白。
