Separation of Biopolymers by Countercurrent Chromatography

本文研究了人血清中三大类脂蛋白、单链结合蛋白（SSB）和大肠杆菌中H-NA的一步纯化，并采用十字轴螺旋行星离心机和两相水相聚合物系统对高、低密度脂蛋白（HDLs、LDLs）进行了分离。在pH9.4条件下，以16%聚乙二醇1000和12.5%磷酸氢二钾组成的聚合物相体系分离效果最好。极低脂蛋白（VLDL）与血清蛋白一起收集。将每个级分浓缩并进行羟基磷灰石层析，以获得三种脂蛋白级分（HDL、LDL和VLDL），其均不含血清蛋白，如白蛋白和球蛋白。用十字轴螺旋管行星离心机从大肠杆菌粗提物中纯化SSB，采用由16%PEG 1000-17%硫酸铵组成的水相聚合物-两相体系。逆流色谱显示两个主要峰，一个峰含有大肠杆菌产生的大部分蛋白质，另一个（组分51）含有SSB。

Three main classes lipoproteins from human serum, one step purification of single stranded binding protein (SSB) and H-NA from E.coli were studied by countercurrent chromatography.High- and low-density lipoproteins (HDLs, LDLs) were fractionated by countercurrent chromatography using the cross-axis coil planet centrifuge using two-phase aqueous polymer systems. The best separations were achieved with a polymer phase system composed of 16% polyethylene glycol 1000 and 12.5% dibasic potassium phosphate at pH 9.4.HDL-LDL fractions were collected from 4 ml of human serum within 3h. Very-low-lipoproteins (VLDLs) were collected together with serum proteins. Each fraction was concentrated and subjected to hydroxyapatite chromatography to obtain three lipoprotein fractions (HDLs, LDLs and VLDLs) all free of serum proteins, such as albumin and globulin. Each lipoprotein was confirmed by agarose gel electrophoresis.SSB purification from crude extracts of E.coli was performed by cross-axis coil planet centrifuge using aqueous polymer-two phase system composed of 16% PEG 1000-17% ammonium sulfate. Countercurrent chromatogram gives two main peaks, one contains most of proteins produced by E.coli, and the other one (fraction 51) contains SSB.SSB was confirmed by SDS-PAGE.