We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.
- Authors
Gutiérrez-Granados, Sonia; Cervera, Laura; Segura, María de las Mercedes; Wölfel, Jens; Gòdia, Francesc
- Abstract
HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 10 VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.
- Subjects
CELL lines; DNA analysis; CELL analysis; PROTEIN analysis; PROTEIN expression
- Publication
Applied Microbiology & Biotechnology, 2016, Vol 100, Issue 9, p3935
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-015-7213-x