Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.
Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain. The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand. Therefore several fragments of this region have been expressed as Flag-fusions in S. cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region. By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway. All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence. Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted. High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases. After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody. The fusion protein concentration in the supernatant was 30 microg/ml. The affinity-purified and deglycosylated protein is a tool for further biochemical and biophysical characterization of SLAM.
Schuster
M
M
Novartis-Forschungsinstitut, Brunnerstrasse 59, 1235 Vienna, Austria.
Wasserbauer
E
E
Aversa
G
G
Jungbauer
A
A
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D007136:Immunoglobulins / Q000235:genetics* / Q000302:isolation & purification; D037102:Lectins; D051379:Mice; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018076:DNA, Complementary; D006023:Glycoproteins; D007136:Immunoglobulins; D037102:Lectins; D011948:Receptors, Antigen, T-Cell; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1