Expression of the extracellular domain of the human immunodeficiency virus type 1 envelope protein and its fusion with beta-galactosidase in Saccharomyces cerevisiae

Abstract

Two envelope glycoprotein gene fragments were cloned from the proviral genome of the HXB2 isolate of human immunodeficiency virus (HIV). For the production of the two domains of the envelope gene product these cloned gene fragments were inserted into an Escherichia coli-yeast inducible shuttle vector fused to the galactokinase (GAL1) promoter. Cell extracts from strains of Saccharomyces cerevisiae harboring these two vectors (pYENV1 and pYENV2) were found to contain a specific protein with a size of 50 kDa when induced by galactose, while the protein could not be detected in extracts from control cells containing only the E. coli-yeast vector in the presence of galactose. Furthermore, another expression plasmid coding for fusion proteins from the majority of the external envelope glycoprotein (gp120) moiety and a large part of the beta-galactosidase was constructed. Antibodies from HIV type 1-positive sera could react with recombinant fusion polypeptides. Transformants could produce this fusion protein to a level of about 1.6% of the total protein content, as deduced from beta-galactosidase activity.

FIG. 1

Detection of recombinant HIV isolate HXB2 envelope polypeptides in S. cerevisiae by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Plasmids pYENV1 and pYENV2 were transformed into S. cerevisiae BJ2168, and transformants were induced by the addition of galactose to 3%. Samples (5 ml) were taken and assayed at different time points. Lanes A to C, BJ2168 carrying pYENV2; lanes D to F, BJ2168 carrying pYENV1; lane J, BJ2168 carrying pYES2. The molecular masses (in kilodaltons [KD]) of standard proteins are shown at the left. The arrows indicate the positions of the env polypeptides.

FIG. 2

Immunogenic reactivity of recombinant fusion polypeptide as tested by dot immunoblotting. Cell lysates with serial protein concentrations prepared from cells containing pYENVG12 (lane A) and pYESGal (lane B) were spotted on the polyvinylidene difluoride membrane and subjected to immunoblot analysis.