Production of endothelin receptor A (ETAR) pseudotyped VLP-s
Expression plasmid construction
For production of ETAR-pseudotyped VLPs, a vector containing two expression cassettes was used. For efficient budding, the expression level of gag protein should be relatively high. Therefore strong CMV promoter containing expression cassette was used for HIV-1 gag protein expression. Various expression cassettes for expression of pseudotyping protein could be used according to nature of the protein. Due to possible toxic effect of ETAR, a weaker hEF1α promoter containing expression cassette was used (Figure 1).
Figure 1. Schematic representation of ETAR-pseudotyped VLP expression vector.
Maintenance sequence (EBV Family of Repeats FR); PyV core origin – murine polyomavirus origin of replication; SV40 pr – SV40 promoter controlling expression of Neo resistance gene; Neo/Km – Neomycin/Kanamycin resistance marker; PolyA – polyadenylation sequence; CMV and hEF1α – promoters of HIV-1 gag or pseudotyping promoter expression cassettes, respectively; Gag – HIV-1 gag protein; ETAR – endothelin A receptor.
Transfection and VLP expression
6x10^6 exponentially growing 293EBNALT75 cells (viability 96%) were transfected with 1 µg of ETAR-VLP expression plasmid using Bio-Rad Gene Pulser electroporator. 72 h after transfection the temperature was decreased to 30°C and the culture was additionally fed. 10 days after the transfection culture was harvested, cell culture supernatant was filtrated and stored at +4°C prior VLP purification. After production VLP expression was analyzed by Western blot analysis using anti HIV-1 gag and anti-ETAR antibodies (Figure 2).
Figure 2. Western-Blot analysis of VLP samples.
Line 1. Size marker; Line 2. ETAR expressionsupernatant; Line 3. ETAR and HIV-1 gag expression supernatant; Line 4. Size marker; Line 5. ETAR-expression supernatant; 6. ETAR and HIV-1 gag expression supernatant. Detection: Lines 2-3 anti-ETAR antibody; Lines 5-6 anti-HIV-1 gag antibody.
Purification and purity analysis
For purification of VLPs, Retro-Concentin solution (System Biosciences) was used according to manufacturer's protocol. After purification VLPs were analyzed by Dynamic Light Scattering using Zetasizer instrument (Malvern) and according software (Figure 3)
| Figure 3. Electron microscopy analysis of HIV-1 gag
VLPs. Dynamic light scattering analysis of HIV-1 gag VLPs. |