Lee K K, Liu P C, Chen Y L
Department of Aquaculture, National Taiwan Ocean University, Keelung.
Electrophoresis. 1999 Nov;20(17):3343-6. doi: 10.1002/(SICI)1522-2683(19991101)20:17<3343::AID-ELPS3343>3.0.CO;2-6.
Electrophoretic characterization of a novel cysteine protease produced by pathogenic luminous Vibrio harveyi, originally isolated from diseased tiger prawn Penaeus monodon in Taiwan, is demonstrated in the present study using native polyacrylamide gel electrophoresis (native PAGE), sodium dodecyl sulfate-PAGE (SDS-PAGE), crossed immunoelectrophoresis (CIE) and isoelectric focusing (IEF) gels. The protease has a pI of 6.4 and exhibits a fast-migrating feature in native-PAGE and CIE gels indicating that it is a negatively charged protease. The protease electrophoresed as a 22 kDa protein band in native- and SDS-PAGE (in SDS - buffer with or without the presence of 2-mercaptoethanol) while it electrophoresed as a 38 kDa protein band in SDS-PAGE when the samples were boiled for 10 min prior to electrophoresis. The results reveal that the enzyme is an SDS-resistant monomeric protease and its high negative charge is not influenced by SDS (detergent) without boiling the sample. The present results are useful in determining proteins of similar nature to this unique cysteine protease.
本研究利用非变性聚丙烯酰胺凝胶电泳（native PAGE）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、交叉免疫电泳（CIE）和等电聚焦（IEF）凝胶，展示了对一种新型半胱氨酸蛋白酶的电泳表征。该蛋白酶最初从台湾患病的斑节对虾中分离出来，由致病性发光哈维氏弧菌产生。该蛋白酶的等电点为6.4，在非变性PAGE和CIE凝胶中呈现快速迁移特征，表明它是一种带负电荷的蛋白酶。在非变性和SDS-PAGE中（在含或不含2-巯基乙醇的SDS缓冲液中），该蛋白酶电泳显示为一条22 kDa的蛋白带，而当样品在电泳前煮沸10分钟时，在SDS-PAGE中它电泳显示为一条38 kDa的蛋白带。结果表明，该酶是一种抗SDS的单体蛋白酶，在不煮沸样品的情况下，其高负电荷不受SDS（去污剂）影响。目前的结果有助于确定与这种独特的半胱氨酸蛋白酶性质相似的蛋白质。
