Smith H, Bron S, Van Ee J, Venema G
J Bacteriol. 1987 Jul;169(7):3321-8. doi: 10.1128/jb.169.7.3321-3328.1987.
To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.
为研究革兰氏阳性菌中分泌蛋白信号肽的多样性和效率，构建了两种质粒载体，用于探测枯草芽孢杆菌中的输出信号编码区。这些载体含有编码细胞外蛋白的基因（地衣芽孢杆菌的α-淀粉酶基因和大肠杆菌的β-内酰胺酶基因），它们缺乏功能性信号序列。通过鸟枪法克隆枯草芽孢杆菌染色体DNA的限制性片段，选择了多种不同的输出编码区。这些区域在枯草芽孢杆菌和大肠杆菌中均有功能。在许多恢复了蛋白质输出的情况下，可以检测到大小增加的细胞内前体蛋白，这些前体蛋白在跨细胞膜转运时被加工成成熟产物。获得输出信号编码区的高频率表明，除了天然信号序列外，许多随机克隆的序列也可以作为输出信号发挥作用。
