title: Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae.
abstract: Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and α-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and α-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (µmol/L) of IP was found to be higher than that of α-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Zihe
authors.initials: Z
authors.lastname: Liu
authors.forename: Keith E J
authors.initials: KE
authors.lastname: Tyo
authors.forename: José L
authors.initials: JL
authors.lastname: Martínez
authors.forename: Dina
authors.initials: D
authors.lastname: Petranovic
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D001709:Biotechnology / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D000078790:Insulin Secretion; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
chemical_list: D007328:Insulin; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000516:alpha-Amylases