Payne M S, Jackson E N
Central Research and Development Department, E. I. du Pont de Nemours & Co., Wilmington, Delaware 19880-0228.
J Bacteriol. 1991 Apr;173(7):2278-82. doi: 10.1128/jb.173.7.2278-2282.1991.
We have constructed a vector designed to facilitate the study of protein secretion in Bacillus subtilis. This vector is based on a translational fusion between the expression elements and signal sequence of Bacillus amyloliquefaciens alkaline protease and the mature coding sequence for Escherichia coli alkaline phosphatase (phoA). We show that export of alkaline phosphatase from B. subtilis depends on a functional signal sequence and that alkaline phosphatase activity depends upon secretion. The vector design facilitates the insertion of heterologous coding sequences between the signal and phoA to generate three-part translational fusions. Such phoA fusions are easily analyzed by monitoring alkaline phosphatase activity on agar plates or in culture supernatants or by immunological detection. Exploitation of this methodology, which has proven to be extremely useful in the study of protein secretion in E. coli, has a variety of applications for studying protein secretion in B. subtilis.
我们构建了一种载体，旨在促进对枯草芽孢杆菌中蛋白质分泌的研究。该载体基于解淀粉芽孢杆菌碱性蛋白酶的表达元件与信号序列以及大肠杆菌碱性磷酸酶（phoA）的成熟编码序列之间的翻译融合。我们表明，枯草芽孢杆菌中碱性磷酸酶的输出取决于功能性信号序列，并且碱性磷酸酶活性取决于分泌。该载体设计便于在信号序列和phoA之间插入异源编码序列，以产生三部分翻译融合体。通过监测琼脂平板上或培养上清液中的碱性磷酸酶活性或通过免疫检测，可以轻松分析此类phoA融合体。这种方法已被证明在大肠杆菌蛋白质分泌研究中非常有用，它在枯草芽孢杆菌蛋白质分泌研究中有多种应用。
