Improved trimerization of JRFL-SOSIP.R6 gp140 by addition of an heterologous trimerization domain. A. Schematic of the SOSIP.R6.IZ design. The clade B JR-FL gp140 (amino acids 31-681) contains several modifications that have been previously described (see Materials and Methods). Trimer formation was further enhanced by insertion of a GCN4-based isoleucine zipper (IZ) to the C-terminus of SOSIP.R6. B. Reducing SDS-PAGE and Blue Native-PAGE analysis of SOSIP.R6 and SOSIP.R6-IZ proteins secreted from transiently transfected 293T cells. Note that no exogenous furin was added in these experiments, therefore the proteins are predominantly (> 90%) uncleaved. C. Gel filtration analysis of SOSIP.R6 and SOSIP.R6-IZ proteins. Concentrated culture supernatants, derived from transiently transfected 293T cells, containing the SOSIP.R6 or SOSIP.R6-IZ proteins were fractionated on a Superose-6 column, followed by analysis by SDS-PAGE and western blot. The elution of standard proteins is indicated.