Catcher-VLPs based on the AP205 coat protein (gene ID 956335) fused to the proprietary catcher sequence were produced in E. coli (45 (link)). The purified S1 was coupled to the catcher-VLPs and formulated as an adjuvanted vaccine as previously described (34 (link)). In short, S1 and catcher-VLPs were mixed in a 1:1 molar ratio in PBS overnight at room temperature. A part of the mix was spun down at 16,000 × g for 2 min to assess the stability of the coupled protein. Equal amounts of pre- and postspin samples were mixed with DTT and were heated prior to analyses by SDS-PAGE. The coupling efficiency was calculated as the percentage of AP205 capsids that had been conjugated to an S1 domain via tag-catcher interactions (76 (link)). The protein bands on the SDS-PAGE gel were analyzed by ImagequantTL software to determine the band intensity. The band intensity of the VLP subunit before the coupling reaction was divided by the equivalent protein band after coupling and was multiplied by 100. After the coupling reaction, the S1-VLP was purified from the remaining coupling mixture. This was loaded onto an OptiPrep step gradient (23%, 29%, and 35%) (Sigma-Aldrich) and was centrifuged at 47,800 rpm for 3.30 h. Buffer exchange was then performed by dialysis in PBS.
Optiprep step gradient
Manufactured by Merck Group
Sourced in United States
Optiprep step gradient is a density gradient medium used for the separation and purification of cells, organelles, and other biological particles. It is a sterile, pyrogen-free, and endotoxin-free solution of iodixanol in water. The Optiprep gradient can be prepared in various step or continuous configurations to suit the specific requirements of the separation process.
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4 protocols using optiprep step gradient
1
Catcher-VLP Vaccine Protocol
2
Pseudovirus and Quasivirus Particle Production
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Pseudovirus (PsV) and Quasivirus (QV) particles were produced by transfecting 293TT cells with HPV16 sheLL plasmids (a gift from the Schiller Lab, NCI, Bethesda, MD, USA) together with either the plasmid for secreted alkaline phosphatase (pSEAP) or cottontail rabbit papillomavirus (CRPV) genome respectively as previously described [23 (link),24 (link),45 (link),46 (link),47 (link)]. Two days post-transfection, cells were collected and lysed using the detergent Brij58 (Sigma-Aldrich, St. Louis, MO, USA). Particles were matured by incubating cell lysates at 37 °C for 24 h and subsequently treating with Benzonase (Sigma-Aldrich, St. Louis, MO, USA) and Plasmid Safe (Epicentre, Madison, WI, USA). Purification of the resulting particles was performed by ultracentrifugation of the lysates at 234,000× g for 3.5 h on an Optiprep step gradient (Sigma-Aldrich, St. Louis, MO, USA). Particles were harvested by puncturing the bottom of the centrifuge tube and collecting fractions of ~250 µL.
3
Acinetobacter Phage AP205 VLP Design
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The Acinetobacter phage, AP205, displaying two SpyTags per VLP subunit, was designed as previously described.31 (link) In brief, the gene encoding the AP205 coat protein (Gene ID: 956335) had the SpyTag peptide sequence (AHIVMVDAYKPTK) fused to both the N- and C-terminus. Each SpyTag was separated from the coat protein by a flexible linker (GSGTAGGGSGS, N-terminus and GTASGGSGGSG, C-terminus). The Spytagged VLP was expressed in Escherichia coli One Shot® BL21 Star™ (DE3) cells (Thermo Scientific) and purified by density gradient ultracentrifugation using an Optiprep™ step gradient (23, 29 and 35%) (Sigma).
4
Production and Purification of Pseudovirions
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PsV were produced in 293TT cells as described [28 (link),29 ]. Briefly, 1.5x107 293TT cells (175cm2 flask) were plated and one day later transfected with 38μg HPV L1 and L2 plasmid(s) and 38μg reporter plasmid. 48 hours later, cells were lysed in PBS with 9.5mM MgCl2, 0.5% (v/v) Triton-X100, 0.25% Ammonium sulfate, 0,01% benzonase (Novagen) and 0,01% Plasmid Safe (Epicentre). For maturation cell lysates were incubated 24 hours at 37°C. Finally, PsV were salt extracted and purified by ultracentrifugation on an Optiprep step gradient (27%, 33%, 39%) (Sigma). Plasmids for codon-optimized L1 and L2 expression of HPV16 (p16shell) and HPV18 (peL1fB and peL2bhb) were provided by J. Schiller, NCI, for HPV45 (p45shell) by J. Dillner, Karolinska Institute, and for HPV39, HPV59, HPV68, HPV70 (all in pVITRO) by R. Roden, Johns Hopkins University.
The reporter plasmid pYSEAP (encoding secreted alkaline phosphatase) was used for in vitro L1 or L2-based PsV assays, and pCLUC (encoding luciferase) for in vitro L2-based PsV assays and in vivo experiments.
The reporter plasmid pYSEAP (encoding secreted alkaline phosphatase) was used for in vitro L1 or L2-based PsV assays, and pCLUC (encoding luciferase) for in vitro L2-based PsV assays and in vivo experiments.
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