高效快速纯化增强型绿色荧光蛋白（EGFP）、增强型黄色荧光蛋白（EYFP）和增强型青色荧光蛋白（ECFP），产率高且纯度高。

Rapid purification of EGFP, EYFP, and ECFP with high yield and purity.

作者信息

McRae Shelley R, Brown Christopher L, Bushell Gillian R

机构信息

Faculty of Science and the Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Qld 4111, Australia.

出版信息

Protein Expr Purif. 2005 May;41(1):121-7. doi: 10.1016/j.pep.2004.12.030.

DOI:10.1016/j.pep.2004.12.030

PMID:15802229

Abstract

Most current high throughput purification procedures for the green fluorescent protein (GFP) suffer from poor yields and low purity. An improved purification procedure that delivers highly pure protein (>95% homogeneity) in high yields (>70% of the initial fluorescent protein content) has been developed. The purification procedure requires only two steps: the cell lysate is heated to 60 degrees C for 4 min in ammonium sulfate and triethylamine, followed by hydrophobic interaction chromatography using isopropanol during the elution phase. The resulting pure product exhibits the same fluorescence profile as the crude sample. This procedure has been demonstrated on three commercial variants of GFP from Aequorea victoria, enhanced green, enhanced yellow, and enhanced cyan fluorescent protein (Becton-Dickinson). The yield and purity of material are superior to other recently described methods.

摘要

目前大多数用于绿色荧光蛋白（GFP）的高通量纯化方法都存在产量低和纯度不高的问题。现已开发出一种改进的纯化方法，该方法能够以高产量（>初始荧光蛋白含量的70%）获得高纯度蛋白质（>95%的均一性）。该纯化方法仅需两步：将细胞裂解物在硫酸铵和三乙胺中加热至60摄氏度4分钟，然后在洗脱阶段使用异丙醇进行疏水相互作用色谱法。所得的纯产品显示出与粗样品相同的荧光谱。该方法已在来自维多利亚水母的三种商业GFP变体上得到验证，即增强型绿色、增强型黄色和增强型青色荧光蛋白（Becton-Dickinson）。该材料的产量和纯度优于其他最近描述的方法。