Weigel L M, Brandt M E, Norgard M V
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Infect Immun. 1992 Apr;60(4):1568-76. doi: 10.1128/iai.60.4.1568-1576.1992.
The 47-kDa lipoprotein is an abundant integral membrane protein and dominant immunogen of Treponema pallidum subsp. pallidum. Previous DNA sequencing of the 47-kDa-lipoprotein gene did not reveal consensus features representative of other bacterial lipoprotein genes; this prompted further analyses with emphasis on elucidation of the N terminus of the molecule. To assist in localizing start signals for the protein, the transcription initiation site for the 47-kDa-antigen gene was determined. RNA isolated from both T. pallidum and recombinant Escherichia coli expressing the 47-kDa antigen was used as a template in reverse transcriptase primer extension. Upon analysis of cDNA products, transcription initiation was localized to one nucleotide in T. pallidum and to two adjacent nucleotides in E. coli. When various primers were used in DNA sequencing reactions for these analyses, a previously undetected nucleotide (G) was found in the purported 5' untranslated region; this altered the upstream reading frame to reveal plausible sites for ribosome binding (GGAGG), translation initiation (GTG start codon), and signal peptidase II processing (Val-Val-Gly-Cys). Discounting acylation, the molecular weight of the mature polypeptide is 45,756 (approximately 46,600 with acylation). To derive nonacylated 47-kDa antigen for further structure-function studies, the 47-kDa-antigen gene was subcloned without acylation signals as a genetic construct encoding a glutathione S-transferase fusion protein; following cleavage with thrombin, the nonacylated 47-kDa protein was hydrophilic rather than amphiphilic but retained its antigenicity when tested against 116 human serum samples from patients with various stages of syphilis.
47 kDa脂蛋白是梅毒螺旋体苍白亚种丰富的整合膜蛋白和主要免疫原。先前对47 kDa脂蛋白基因的DNA测序未揭示出代表其他细菌脂蛋白基因的共有特征；这促使人们进一步分析，重点是阐明该分子的N端。为了帮助定位该蛋白的起始信号，确定了47 kDa抗原基因的转录起始位点。从梅毒螺旋体和表达47 kDa抗原的重组大肠杆菌中分离的RNA用作逆转录酶引物延伸的模板。分析cDNA产物后，转录起始定位在梅毒螺旋体中的一个核苷酸和大肠杆菌中的两个相邻核苷酸处。当在这些分析的DNA测序反应中使用各种引物时，在假定的5'非翻译区发现了一个先前未检测到的核苷酸（G）；这改变了上游阅读框，从而揭示了核糖体结合（GGAGG）、翻译起始（GTG起始密码子）和信号肽酶II加工（Val-Val-Gly-Cys）的合理位点。不考虑酰化作用，成熟多肽的分子量为45,756（酰化后约为46,600）。为了获得用于进一步结构功能研究的非酰化47 kDa抗原，将47 kDa抗原基因作为编码谷胱甘肽S-转移酶融合蛋白的基因构建体进行亚克隆，去除了酰化信号；用凝血酶切割后，非酰化的47 kDa蛋白是亲水性而非两亲性的，但在针对116份来自梅毒各阶段患者的人血清样本进行测试时，保留了其抗原性。
