Hu H M, Shih K N, Lo S J
Institute of Microbiology and Immunology, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC.
J Virol Methods. 1996 Jun;60(1):39-46. doi: 10.1016/0166-0934(96)02029-0.
The in vitro transcription and translation coupling system has been demonstrated to be a powerful method of characterizing protein encoded by a cloned gene. Two cDNA constructs coding hepatitis D viral (HDV) antigen, small (S) and large (L) DAg, respectively, were subjected to in vitro transcription and translation to examine multimer formation ability. By using 2-D-SDS-PAGE (non-reducing and reducing) analysis, two novel characteristics of the LDAg were found: (i) the forming of a homodimer and (ii) the formation of a complex with an unidentified 11-kDa protein via a disulfide bond. These features were neither found in SDAg nor in a cysteine-negative mutant of LDAg. Based on the fact of isoprenylation occurring at the sole cysteine residue of LDAg, it is suggested that the formation of a disulfide bond of LDAg might be involved in a transition toward isoprenylation.
体外转录与翻译偶联系统已被证明是表征克隆基因所编码蛋白质的一种强大方法。分别编码丁型肝炎病毒（HDV）抗原小（S）和大（L）DAg的两种cDNA构建体进行体外转录和翻译，以检测多聚体形成能力。通过二维SDS-PAGE（非还原和还原）分析，发现了LDAg的两个新特性：（i）形成同二聚体；（ii）通过二硫键与一种未鉴定的11 kDa蛋白质形成复合物。这些特性在SDAg中未发现，在LDAg的半胱氨酸阴性突变体中也未发现。基于LDAg唯一的半胱氨酸残基发生异戊二烯化这一事实，提示LDAg二硫键的形成可能参与向异戊二烯化的转变。
