棒状链霉菌蛋白质样品提取方法及双向凝胶电泳条件的优化

Establishment and optimization of protein extraction and two-dimensional gel electrophoresis(2-DE) method for Streptomyces clavuligerus proteome

针对基于工业用复合培养基的棒状链霉菌（Streptomyces clavuligerus）发酵液杂质含量高特性，对蛋白质样品制备、第一向等电聚焦和第二向SDS．PAGE电泳相关的主要实验条件进行了研究和优化。所得最适条件为：采用液氮研磨方法破碎细胞，用10％的TCA／丙酮沉淀法除盐，用含有7mol／L尿素，2mol／L硫脲，2％CHAPS，20mmol／L DTT，0．5％IPG缓冲液（pH3～10）的溶胀液重悬蛋白，应用pH梯度为4—7，长度为24cm的固定化胶条进行第一向等电聚焦，浓度为12．5％的聚丙烯酰胺凝胶进行第二向SDS—PAGE，考马斯亮蓝R-250染色，上样量为600μg。对获得的双向电泳图像运用Image Master 2D Platinum软件进行分析表明成功建立了重复性好的基于复合培养基的棒状链霉菌蛋白质样品制备和双向电泳方法，为工业规模克拉维酸发酵过程蛋白质组研究奠定了基础。

In the present paper the protocols for soluble cellular protein extraction in Streptomyces clavuligerus grown on complex medium and two-dimensional gel eleetrophoresis （2-DE） were investigated and optimized. The main results were as followed： grinding with liquid nitrogen to disrupt cells, 10% TCA/acetone precipitation to make protein desalted, using 0.5% IPG buffer （pH 3 - 10）, 7 mol/L urea, 2 mol/L thiourea, 2% CHAPS, 20 mmol/L DTT to redissolve protein sample, IEF with immobilized pH gradients （4 -7） gel as the first dimension electrophoresis, 12.5% SDS-PAGE as the second, sample amount 600 μg, stained with Coomassie blue. The fine 2-DE pattern of Streptomyces clavuligerus cellular protein with high resolution and reproducibility was obtained.