Partial characterization of the 30 kD Ig-binding protein from Pseudomonas maltophilia.
Partial characterization of the 30 kD Ig-binding protein from Pseudomonas maltophilia.
复制标题
嗜麦芽假单胞菌 30 kD Ig 结合蛋白的部分表征。
DOI：
10.1016/0006-291x(92)91841-d
复制
发表时间：
1992
影响因子：
3.1
通讯作者：
Odell,WD
中科院分区：
文献类型：
--
作者：
Grover,S;Odell,WD
我们先前已经证明嗜麦芽假单胞菌(ATCC 13637)具有一个30 kDa的细胞壁蛋白，通过其Fc区结合不同亚类的Ig G和Ig A。用木瓜酶溶解蛋白，用溴化氰活化的琼脂糖珠与人免疫球蛋白偶联亲和层析纯化蛋白。洗脱液在12%的聚丙烯酰胺凝胶上变性，Western印迹分析鉴定免疫活性条带，第二凝胶考马斯亮蓝染色。亲和纯化的洗脱液在一维15%聚丙烯酰胺凝胶上进行电泳层析，考马斯亮蓝染色。切下兴趣蛋白条带。用金黄色葡萄球菌V-8酶原位消化蛋白条带。在15%的聚丙烯酰胺凝胶上进行第二次一维分离，然后电印迹到聚偏二氟乙烯膜上。条带通过考马斯亮蓝染色可视化，切下，并使用自动气相测序仪进行测序。以类似的方式测定最低氨基酸组成。通过上述方法，我们获得了部分N-末端氨基酸序列数据。
We have previously demonstrated that Pseudomonas maltophilia (ATCC 13637) possess a 30 kDa cell wall protein which binds various subclasses of IgG's and IgA by their Fc region. The protein was solubilized by papain and purified by affinity chromatography on cyanogen bromide activated sepharose beads conjugated with human IgG. The eluent was electrophoresed on a 12% polyacrylamide gel under denaturing conditions, and the immunoactive bands identified by Western blot analysis, a second gel was stained with Coomassie blue. The affinity purified eluent was electrophoresed on a one-dimensional 15% polyacrylamide gel and stained with Coomassie blue. The protein band of interst was cut. The protein band was then digested in situ with Staphylococcus aureus V-8 protease. The peptide bands were separated by electrophoresis on a second one dimensional 15% polyacrylamide gel and then electroblotted onto a polyvinylidine difluoride membrane. The bands were visualized by staining with Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Minimal amino acid composition was determined in a similar fashion. We have thus obtained partial N-terminal amino acid sequence data from the above method.