基于尺寸排阻色谱法的蛋白质复性过程中样品应用和洗脱的考量。

Considerations of sample application and elution during size-exclusion chromatography-based protein refolding.

作者信息

Batas B, Chaudhuri J B

机构信息

Department of Chemical Engineering, University of Bath, Claverton Down, UK.

出版信息

J Chromatogr A. 1999 Dec 24;864(2):229-36. doi: 10.1016/s0021-9673(99)01030-4.

DOI:10.1016/s0021-9673(99)01030-4

PMID:10669290

Abstract

A mechanism for size-exclusion chromatography-based protein refolding is described. The model considers the steps of loading the denatured protein onto a gel filtration column, and protein elution. The model predictions are compared with results of refolding lysozyme (10 and 20 mg/ml) using Superdex 75 HR. The main collapse in protein structure occurred immediately after loading, where the partition coefficient of unfolded lysozyme increased from 0.1 to 0.48 for the partially folded molecule. Use of a refolding buffer as the mobile phase resulted in complete refolding of lysozyme; this eluted at an elution volume of 15.6 ml with a final partition coefficient of 0.54. The model predicted the elution volume of refolded lysozyme at 19.3 ml.

摘要

描述了一种基于尺寸排阻色谱的蛋白质复性机制。该模型考虑了将变性蛋白质加载到凝胶过滤柱上的步骤以及蛋白质洗脱步骤。将模型预测结果与使用Superdex 75 HR复性溶菌酶（10和20 mg/ml）的结果进行了比较。蛋白质结构的主要折叠发生在加载后立即，在此处，未折叠溶菌酶的分配系数从0.1增加到0.48，形成部分折叠分子。使用复性缓冲液作为流动相导致溶菌酶完全复性；其在15.6 ml的洗脱体积处洗脱，最终分配系数为0.54。模型预测复性溶菌酶的洗脱体积为19.3 ml。