Introduction: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. Objectives: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. Methods: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. Results: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. Conclusion: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.

中文翻译：

---

使用内含肽技术进行 B19V VP1u 结构域分割进行结构研究的可行性

简介：细小病毒B19（B19V）是一种人类病原体，B19V的次要衣壳蛋白拥有一个独特的N末端，称为VP1u，在病毒的生命周期中发挥着至关重要的作用。目的：本研究的目的是开发一种使用内含肽技术对 B19 VP1u 进行结构域分割的方法，特别是其受体结合结构域 (RBD) 和磷脂酶 A2 (PLA2) 结构域。方法：VP1u 的 RBD 和 PLA2 结构域均与来自点状发菜的 DnaE 分裂内含肽融合。这些前体蛋白均在大肠杆菌中表达。将纯化的前体以等摩尔比混合，形成全长 VP1u。此外，圆二色性 (CD) 光谱和 PLA2 测定用于探测新形成的蛋白质的结构和活性。结果：全长 VP1u 的 CD 谱证实了蛋白质的二级结构，而 PLA2 测定表明酶活性的破坏最小。结论：该方法允许选择性地将 NMR 活性同位素掺入任一 VP1u 结构域，从而减少 NMR 结构测定研究中的信号重叠。