嗜酸氧化亚铁硫杆菌ATCC23270周质蛋白的选择性提取及差异表达

Selective extraction and differential electrophoregrams analysis of periplasmic proteins of Acidithiobacillus ferrooxidans ATCC23270

通过考察萃取时间、高渗液与低渗液的摩尔比以及总的渗透液体积对细胞质苹果酸脱氢酶的比活性(相对于细胞蛋白质总量)的影响,建立选择性提取Acidithiobacillus ferrooxidans ATCC23270周质蛋白的渗透休克实验方法;结合透射电镜和双向电泳实验方法,分析渗透处理前后细胞的形态和不同生长期的细胞周质空间蛋白表达差异。研究结果表明,合适的渗透休克实验条件为:细胞质量为0.1g(湿重),渗透液的总体积为10mL,渗透液的作用时间为15min,高渗液与低渗液的摩尔比为1:1;在高渗环境下,细胞收缩,周质空间变小;在低渗液环境下,细胞周质空间膨胀,外膜被胀破,周质空间内蛋白释放出来;双向电泳显示周质蛋白总体上以小相对分子质量蛋白为主,在不同的生长时期内的周质蛋白表达存在差异。

The selective extraction of periplasmic proteins by osmotic shock was investigated. The effects of the extraction efficiency and the pollution from the cytoplasmic proteins were tested and monitored, respectively, in terms of the specific activity of malate dehydrogenase （MDH）. And the morphology and differential expression of the periplasmic proteins of Acidithiobacillusferrooxidans were analyzed by using TEM and 2-D electrophoresis. The results show that the preferable schema of osmotic shock is founded as follows： the wet mass of cell is 0.1 g, the molar ratios of hypertonic saline to hypotonic saline is 1：1, the volume of the osmotic solvent is 10 mL and the extracting time is 15 rain. The TEM images show that under the treatment from hypertonic to hypotonic environment, the cell outer membrane is selectively burst, which makes the periplasmic space protein released. The 2-D electrophoregrams display the most of periplasmic proteins are small molecular weight protein and cells growth in different phase present difference in periplasmic proteins expression.