Trimeric HIV-1 envelope glycoproteins (Env) are now being evaluated instead of monomeric gp120 as vaccine antigens because they mimic more closely the spikes expressed on the surface of virions. Thus, it can be argued that trimers have a more native structure than gp120, so might be superior at raising neutralizing antibodies. One approach to making Env trimers is to ensure that they are cleaved at the gp120 gp41 border, but stabilized by other, engineered substitutions such as intra-subunit disulfide bonds (SOS and SOSIP gp140 proteins). However, the production of properly folded, cleaved trimers is complicated by the requirement for co-expression of the exogenous protease furin, to facilitate the efficient processing oft the gp120-gp41 cleavage site. Also, yields of purified trimeric SOSIP gp140 proteins are usually moderate and for scale-up procedures the cost of transfection reagents becomes an important economical factor. Here, we assess the optimal culture conditions for the transient expression of these complex proteins. We found that the use of linear polyethylenimine 25 kDa (PEI25k) as a transfection aid was a cost-efficient. economical alternative to several commercially available products. By using PEI25k and an optimized plasmid:furin ratio, we could express proteolytically mature, trimeric Env vaccine antigens at levels high enough for use in immunization or structural studies. We also show that the same transfection method can be used to generate infectious pseudoviruses. (c) 2006 Elsevier Inc. All rights reserved.