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Expression and Secretion of Beta‐Galactosidase in Saccharomyces Cerevisiae Using the Signal Sequences of Ggpi, the Major Yeast Glycosylphosphatidylinositol‐Containing Protein
Author(s) -
Pignatelli Raffaella,
Vai Marina,
Alberghina Lilia,
Popolo Laura
Publication year - 1998
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1998.tb01378.x
Subject(s) - endoplasmic reticulum , secretory pathway , secretory protein , signal peptide , saccharomyces cerevisiae , secretion , biology , biochemistry , fusion protein , glycoprotein , stim1 , cytoplasm , signal peptidase , glycosylation , yeast , microbiology and biotechnology , golgi apparatus , peptide sequence , gene , recombinant dna
New secretory signals and strategies can be attempted to improve the secretion of heterologous proteins of biotechnological interest which encounter difficulties being exported in yeast. The GGP1 g ene of Saccharomyces cerevisiae codes for a 125 kDa glycoprotein transported through the secretory pathway and anchored to the plasma membrane by means of a glycosylphosphatidylinositol. The regions coding for the secretory signal or also for the first 46 amino acids were tested for efficiency in secretion by fusion to the lacZ gene of Escherichia coli resulting in the synthesis of the endoplasmic reticulum‐targeted 1–22‐ and 1–68‐Ggp1p/ b‐gal hybrids. A cytoplasmic form was also examined. The 1‐22bgal is partially transported to the cell surface and in the medium in an unglycosylated form. The 1–68 bgal is completely retained in the intracellular membranes and is N‐glycosylated in the Ggp1p moiety. The amount of hybrid protein produced is similar and independent from its targeted site, suggesting that translocation through endoplasmic reticulum is not a limiting step, whereas the amount of active enzyme is from 50 to 80% lower for the endoplasmic reticulum forms compared with the cytoplasmic form. BiP/Kar2p putative precursor is accumulated in cells expressing the endoplasmic reticulum‐targeted forms but not in those producing the cytosolic b‐galactosidase or overexpressing an endogenous secretory protein. Thus, glycosylation and abnormal folding rather than overexpression are among the factors responsible for the decreased activity and exit of b‐galactosidase from the endoplasmic reticulum and for induction of BiP. The results obtained indicate that the sole secretory signal of Ggp1p is suitable to drive secretion of foreign products with complex folding and point to the importance of the endoplasmic reticulum quality control in the secretion of heterologous proteins in yeast.