title: Genetic immobilization of RNase Rny1p at the Saccharomyces cerevisiae cell surface.
abstract: Genetic immobilization of the yeast RNase Rny1p was performed by creating a hybrid protein containing the signal sequence of the S. cerevisiae cell wall protein Ccw12p followed by the catalytic part of the Rny1p (amino acids 19 to 293) and additionally 73 amino acids of the Ccw12p including the GPI-anchoring signal. The construct was expressed in S. cerevisiae VMY5678 and the hybrid protein was secreted through the plasma membrane and incorporated into the cell wall through GPI-anchoring in the same way as the Ccw12p. Thus, it could be released from the wall by β-1,3-glucanase. It retained RNase activity with the optimal pH of about 9 and the optimal temperature at 60°C. It was significantly more stable than the wild type enzyme and retained activity at 50°C for at least 6 hours; at 60°C it maintained full activity for at least 4 h, and at 70°C it lost activity in about 2 h. No DNase activity of the Rny1/Ccw12p was detected. Yeast cells expressing the hybrid protein were successfully used instead of RNase A in a standard procedure for yeast chromosomal DNA preparation with the advantage of quick and easy quantitative removal of the RNase activity from the reaction mixture.
authors.affiliation: Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia. rteparic@pbf.hr
authors.forename: Renata
authors.initials: R
authors.lastname: Teparić
authors.forename: Blanka
authors.initials: B
authors.lastname: Didak
authors.forename: Elena
authors.initials: E
authors.lastname: Ščulac
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mrša
mesh_terms: D061505:Cell Surface Display Techniques / Q000379:methods*; D002473:Cell Wall / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D060847:Metabolic Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012260:Ribonucleases; C540806:Rny1 protein, S cerevisiae