摘要
为建立从Cohn′s组分IV沉淀中分离纯化α1-抗胰蛋白酶(α1-antitrypsin,α1-AT)的技术路线,以Cohn′s组分IV为原料,利用还原剂1,4-二硫苏糖醇(DTT)、气相二氧化硅等处理,再经压滤、离子交换层析和疏水层析提取α1-AT。用SDS-PAGE及合成基质法检测α1-AT的纯度以及生物活性。结果可见,Cohn组分IV沉淀中α1-AT占蛋白总量的11%～13%,经该工艺提取的α1-抗胰蛋白酶纯度为97.6%,疏水层析后α1-AT的收获率为21%,比活为每毫克总蛋白中含0.54mg功能活性α1-AT。可见用此方法从Cohn组分IV沉淀中可制备高纯度的α1-AT。
In order to establish the technical route to isolate and purify α1-antitrypsin （α1-AT） from Cohn＇s component Ⅳ precipitate, the Cohn＇s component Ⅳ paste containing α1-AT was suspended in a buffer. And it was treated with dithiothreitol （DTT） and fumed silica. The suspension was then filtered. The α1-AT final filtrate was applied directly onto ion exchange chromatography （IEC） column and a hydrophobic interaction chromatography （HIC） column. The activity of α1-AT was detected by peptide chromogenic substrate method. The purity of α1-AT was determined by SDS-PAGE and band scanning method. The results showed that α1-AT had 11 - 13% of the total protein in Cohn＇s component Ⅳ. The purity of α1-AT extracted with this technology was 97.6%. The overall recovery of α1-AT through HIC was 21% , and the specific activity was 0.54 functional α1-AT per mg protein. Therefore, these methods could prepare highly pure α1-AT from Cohn＇s component Ⅳ precipitate.
出处
《微生物学杂志》
CAS
CSCD
2008年第1期28-31,共4页
Journal of Microbiology
基金
上海市科学技术委员会科研计划项目(05XDB1419)
关键词
α1
抗胰蛋白酶
Cohn’s组分Ⅳ
还原剂
离子交换层析
疏水层析
α1-antitrypsin
Cohn＇s component Ⅳ
reducing agent
ion exchange chromatography （IEC）
hydrophobic interaction chromatography （HIC）
