Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of botulinum neurotoxin in Saccharomycescerevisiae

Effective expression of the HIV-1 core protein Gag-p55 was obtained in Sach aromyces cerevisiae under control of the inducible UASgal/CYC1 promoter as a translational fusion with the prion-forming NM domain of the translation terminator Sup35p (eRF3) of S. cerevisiae, where only poor expression of th e original-type Cag-p55 was observed. A deletion within the Sup35NM prion-f orming domain altering Sup35-associated [PST] inheritance did not compromis e expression of the Sup35NM Gag-p55 fusion protein. Therefore, either the m echanism of this phenomenon is not directly related to the effect of Sup35p prion-formation or the modified protein maintains residual prion-forming a bilities. The recombinant Sup35p-Gag-p55 protein was quite stable under boi ling in an alkali/sodium dodecyl sulfate (SDS) solution and completely reta ined its antigenic properties. Moreover, 10-min boiling of the native yeast cells in this solution allowed immediate inhibition of lysosomal and other yeast proteases, responsible for autolysis of many natural and recombinant proteins. The use of this method of preliminary enrichment for the recombi nant fusion protein Sup35p-Gag-p55 with the SDS-alkaline extraction could b e useful for yeast heterologous expression and purification of other of ins oluble and unstable proteins. A translational fusion with the NM domain of Sup35p was also used to produce another poorly soluble protein, the L-chain of botulinum exotoxin A, in S. cerevisiae. When the Sup35p fragment was re moved from the recombinant construct encoding a fused Sup35/BoNT protein, a dramatic drop in both transformation efficiency and growth rate of transfo rmants was shown. (C) 2001 Editions scientifiques et medicales Elsevier SAS .