Simple and efficient ultrafiltration method for purification of rotavirus VP6 oligomeric proteins

Abstract

Bacterial endotoxins, DNA, live viruses, and viral proteins derived from bacterial and baculovirus (BV) expression vectors employed in recombinant protein production contaminate the final product. Density gradient centrifugation is commonly used to partially purify oligomeric proteins, but impurities from the expression system still remain. We describe a simple and rapid ultrafiltration method for final purification of rotavirus VP6 oligomeric nanotubes and nanospheres. Contamination originating from the BV vector used in VP6 production was undetectable. The method is highly efficient, fast and inexpensive and can be used for a small-scale laboratory purification of VP6 protein to replace technically demanding multi-step chromatographic procedures.

Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co-delivered with norovirus VLPs

“…GQ477131) used for immunizations of animals were highly purified with multi‐step chromatographic procedures or various steps of ultrafiltration, as described previously . The purified rVP6 was assembled into nanotubes in phosphate‐buffered saline (PBS) at pH 7·3–7·5 (Lonza, Verviers, Belgium) or nanospheres in a 50 mM sodium acetate buffer with 130 mM NaCl, pH 4·82 . The concentration of the proteins was determined using Pierce BCA protein assay (Thermo Scientific, Waltham, MA, USA).…”

“…The concentration of the proteins was determined using Pierce BCA protein assay (Thermo Scientific, Waltham, MA, USA). The purity of the proteins was verified by Quant‐it dsDNA Broad‐Range Assay Kit (Invitrogen, Carlsbad, CA, USA; < 10 ng dsDNA/10 μg of protein), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE), BacPAK RapidTiter Kit [Clontech Laboratories, Mountain View, CA, USA; 0 plaque‐forming units (pfu) live BV/ml] and limulus amebocyte lysate assay (Lonza, < 0·1 endotoxin units/100 μg of protein), as described in detail elsewhere . The VLPs and oligomeric rVP6 nanostructures used for immunizations were confirmed by negative‐staining transmission electron microscopy (TEM) using an FEI Tecnai F12 (Philips Electron Optics, Eindhoven, the Netherlands) after negative staining with 3% uranyl acetate pH 4·6 for protein morphology and integrity (Fig.…”

Rotavirus VP6 Adjuvant Effect on Norovirus GII.4 Virus-Like Particle Uptake and Presentation by Bone Marrow-Derived Dendritic Cells In Vitro and In Vivo

“…GQ477131) were produced in a baculovirus-insect cell expression system and purified through sucrose-gradients as previously described [14,38]. Final purification was conducted by consecutive ultrafiltrations containing dissociation and reconstitution steps as described earlier for RV VP6 [39]. Briefly, the structure of VP6 nanotubules was dissociated in 80 mM sodium acetate (pH 3.0, overnight at +4) and filtrated using Vivaspin 300K centrifugal filter devices (Sartorius AG, Frankfurt, Germany) to remove large impurities.…”

“…The final products were characterized as described earlier [39,41]. Accordingly, the protein concentration was measured using a BCA protein assay kit (Pierce), and morphology of purified RV VP6 nanotubes and NoV VLPs were observed using electron microscopy (EM) ( Figure 1).…”

Rotavirus VP6 as an Adjuvant for Bivalent Norovirus Vaccine Produced in Nicotiana benthamiana

“…Furthermore, RV VP6 displays a high degree of conservation among group A RVs that cause >90% of human RV infections [13]. VP6 forms different oligomeric nanostructures such as nanotubes and nanospheres in vitro, depending on conditions such as pH and ionic strength [14]. VP6 induced immune response has been shown to protect from homologous and heterologous RV infection in animal models [15,16,17,18].…”