蛋白质制备色谱的研究

Preparative High Performance Liquid Chromatography for Proteins

中文关键词:  离子交换色谱  反相液相色谱  顶替色谱  蛋白质

英文关键词:ion exchange chromatography  reversed phase chromatography  displacement chromatography  protein

中文摘要:

      研究了蛋白质纯化制备的二种运行模式.其一,在色谱柱超载下纯化制备蛋白质模式,并成功地用离子交换色谱纯化制备了大豆中的胰蛋白酶,用反相液相色谱纯化了细胞色素C;其二,用溶质溶质顶替色谱纯化制备了核糖核酸酶-A,并对顶替色谱过程中诸参数进行了讨论和选择.

英文摘要:

      Two preparative HPLC model for proteins are discribed. (1) Under condition of volume overloaded on column, the preparative ion exchange and reversed phase chromatography for proteins on column, the volume of the injected sample may be so large that eluted peaks are significantly wider than those from analytical sample. Although the solute concentration is lower, but we still can get more pure protein. First the preparative ion exchange chromatography was prepared trypsin inhibitor from crude soybean by Qme-PEI-si-200(30 μm), under condition of volume overloaded on column. The column were eluted with a gradient from 0.01 mol/L Tris-HCl pH 8 to 0.5 mol/L NaCl pH 8, 40 min, at flowrate of 50 mL/min, 30 min, fractions were collected (Fig. 1,2). Second, the cytochrome C from mix proteins was pured on preparative RP HPLC by using SynChropak C₈, the column were eluted with a gradient from 0% to 80% isopropyl alcohol, containing 0.1% TFA pH 2, at flowrate of 1.0 mL/min 15 min, fractions were collected(Fig. 3). (2) Solute-solute displacement chromatography for proteins. This study deals with of Rnase by RSA-Rnase displacement on reversed phase chromatography. The amount of displacer, volume of displacer, flowrate of column and displacement effect on the gradient elution RP HPLC with Vydac C₄(100×4.5 mm) column is discribed. The optimum displacement and high yield has been obtained.