P15.21
Background: Transient protein expression is a method of choice to produce recombinant HIV-1 proteins for the quick assessment of their structural characteristics. Large-scale transient protein expression in mammalian systems such as HEK293 or CHO cells allows obtaining milligram quantities of eukaryotic viral proteins and antibodies with a relatively high-throughput which require accurate post-translational modifications for proper folding and/or a part of large multimeric complexes.
Methods: We developed the large-scale transient protein production platform in HEK293 cells for rapid production of the milligram quantities of soluble HIV-1 immunogens and monoclonal antibodies sufficient to facilitate the structural studies of HIV-1 sites of vulnerability and antibodies.
Results: Large-scale transient transfection platform provided the means to obtain mg quantities of HIV-1 immunogens and antibodies with high degree of variability which allowed us to perform X-ray structural characterization of HIV-1 viral spike and its sites of vulnerability. Crystal structures of soluble HIV-1 envelope proteins provided atomic-level details on functional constraints, conformational flexibility and antibodies binding sites.
Conclusions: Rapid and efficient transient production of a number of difficult-to-express HIV-1 envelope proteins and antibodies not only facilitates HIV-1 structural studies to delineate the atomic level details, flexibility and conformational constraints of the viral spike but also allows to design and assess structurally stabilized HIV-1 immunogens which altogether establishes the integrative approach en route to vaccine development.
