核心组蛋白及染色质组装相关因子的分离、表达及纯化

Isolation,expression and purification of core histones and chromatin assembly associated proteins

目的为制备体外组装染色质所需的核心组蛋白及蛋白因子。方法应用密度梯度离心和羟基磷灰石柱分离小牛胸腺细胞核心组蛋白H2A、H2B、H3及H4;应用昆虫表达体系表达了染色质组装相关子NAP-1、ACF(包括Acf-1和ISWI两个同时表达的组分),并用离子交换或亲和层析方法进行了纯化。结果本实验报道了制备几种核内蛋白因子的表达与纯化方法和条件,为体外组装染色质模板提供了必要基础。

Objective Core histones H2A, H2B, H3 and H4 were extracted from calf thymus, fractionated on sucrose density gradient ultracentrifugation, and further purified by hydroxyapatite. Methods Chromatin assembly protein factors, NAP-1 （Nucleosome assembly protein-l）, Acf-1 （ATP utilizing chromatin assembly and remodeling factor-l） and ISWI （Imitation Switch） were expressed in Sf9 cells using baculovirus expression system and purified with affinity or ionexchange chromatography techniques. Results The work has provided expression and purification procedures of the nuclear proteins that are required for assembly of chromatin template in vitro.