Expression And Purification Of Murine Polyomavirus Virus-like Particle

Virus-like particle (VLP) of murine polyomavirus (MPV) is a hollownanoparticle that self-assembles from viral structural protein, VP1. It is noninfectiousdue to the lack of genetic material, and thus has great potential in the development ofvaccines, gene therapy, drug delivery, and biomaterials, etc. MPV VLP self-assemblesfrom72capsomeres (Cap), each consisting of a pentamer architecture of five VP1proteins. However, the utilization of thrombin in the expression and purification ofVP1caused high cost and complicated procedures, leading to limited application ofVLP. Therefore, efforts should be devoted to develop novel low-cost expression andpurification method, as shown below in two parts.In part I, intein-mediated expression and purification of VP1was developed. Theself-cleavage feature of intein was utilized by inserting the gene segment of Ssp DnaBintein between VP1and GST to construct a recombinant plasmidpGEX-4T-1-intein-VP1, which was then transformed into E.coli to express fusionprotein GST-intein-VP1. Thereafter, affinity chromatography was used to purify thefusion protein. Temperature and pH in the column were changed to the optimizedcleavage condition (pH7.0,25℃,36h) to induce the self-cleavage of intein. Thus,GST-intein tag was left in the column and VP1was separated. The one-steppurification of VP1was obtained with the production of4mg/(L culture). PurifiedVP1was observed to self-assemble in vitro into VLP through two-step dilutionmethod. The structure of VLP were confirmed consistent with natural MPV VLP,indicating that the VP1possess native structure.In part II, DWDLRLLYC was then experimentally validated by a one-steppurification with the octapeptide. Purification of Cap from cell culture supernatantexpressed by genetic engineering E.coli cells was examined, using affinitychromatography with an ligand N-DWDLRLLYC immobilized on ThiopropylSepharose6B (Pep-6B), where N-DWDLRLLYC is a high affinity ligand of Capfound in our group using a biomimetic design strategy. Almost all Caps in the cellculture supernatant bind to the Pep-6B but cannot be adsorbed to the blank resin, demonstrating that Pep-6B can selectively recognize Cap from a heterogeneousmixture of proteins. Moreover, the purity of VP1was significantly improved from15.6%to70.1%by a one-step purification in affinity chromatography and therecovery of VP1was47±3%. The purified Caps were observed to self-assemble intoVLP with consistent structure of authentic MPV.Therefore, a low cost expression and purification process of VP1is successfullydeveloped, which would be helpful for high-efficiency production and application ofVLP.