Hansen E B, Pedersen P E, Schouls L M, Severin E, van Embden J D
J Bacteriol. 1985 Jun;162(3):1227-37. doi: 10.1128/jb.162.3.1227-1237.1985.
A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start from at least two different treponemal promoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coli, we concluded that a similar proteolytic processing takes place in both E. coli and T. pallidum. Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent than tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front of the structural genes. The nucleotide sequence of the T. pallidum tmpA gene was established. This is the first T. pallidum gene sequenced. Codon usage and the nature of transcriptional and translational signals are discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a signal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having beta-galactosidase enzyme activity as well as TmpA epitopes. The enzyme-linked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage lambda were used. In this case the protein produced was a sandwich protein consisting of 21 amino acids of the lambda cro protein, 204 amino acids of the T. pallidum TmpA protein, and 1,020 amino acids of the E. coli lambda-galactosidase. The potential use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed.
本文描述了梅毒螺旋体DNA先前克隆的5.5千碱基片段的详细物理图谱和遗传图谱。该片段在大肠杆菌中表达了两种与细胞膜相关的蛋白质。这些梅毒螺旋体膜蛋白的结构基因tmpA和tmpB是协调表达的，并且在大肠杆菌中的转录可以从至少两个不同的梅毒螺旋体启动子开始。tmpA和tmpB蛋白是体内从前体蛋白进行蛋白水解切割的产物，前体蛋白分别比成熟蛋白大2和4千道尔顿。由于梅毒螺旋体中产生的相应蛋白的大小与大肠杆菌中成熟膜蛋白的大小相同，我们得出结论，在大肠杆菌和梅毒螺旋体中都发生了类似的蛋白水解加工。尽管tmpA和tmpB受相同转录信号的控制，但tmpB的表达程度高于tmpA，并且只有tmpB产物可以通过将左λ启动子置于结构基因之前而过量产生。确定了梅毒螺旋体tmpA基因的核苷酸序列。这是第一个测序的梅毒螺旋体基因。讨论了密码子使用以及转录和翻译信号的性质。推导的氨基酸序列表明存在信号肽特征性的序列。该序列信息允许构建编码具有β-半乳糖苷酶活性以及TmpA表位的蛋白质的杂合基因。当使用来自大肠杆菌噬菌体λ的转录和翻译信号时，酶联抗原在大肠杆菌中高水平表达。在这种情况下，产生的蛋白质是一种夹心蛋白，由λ cro蛋白的21个氨基酸、梅毒螺旋体TmpA蛋白的204个氨基酸和大肠杆菌λ-半乳糖苷酶的1020个氨基酸组成。讨论了这种酶联抗原在梅毒血清学诊断中的潜在用途。
