Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies

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.

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.

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.

The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA₂ assay indicated minimal disruption in enzymatic activity.

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.