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 e xpression products can be purified up to >90% in a single step. The overexp ression and secretion of the transmembrane glycoprotein signaling lymphocyt ic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobul in superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span tr ansmembrane and a cytoplasmatic domain. The extracellular part may be relev ant for stimulation studies in vitro since SLAM is a high-affinity self-lig and. Therefore several fragments of this region have been expressed as Flag -fusions in S, cerevisiae: a full-length fragment containing the transmembr ane region and the autologous signal sequence, another without the transmem brane 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 i nserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha -leade r 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 fr agment without autologous signal sequence and transmembrane domain could be efficiently secreted, High-mannose glycosylation was analyzed by lectin ma pping and digestion with specific glycosidases. After enzyme treatment, a s ingle band product with the theoretical size could be detected and identifi ed as SLAM by a specific monoclonal antibody. The fusion protein concentrat ion in the supernatant was 30 mug/ml. The affinity-purified and deglycosyla ted protein is a tool for further biochemical and biophysical characterizat ion of SLAM. (C) 2001 Academic Press.