| Autor: |
Pignatelli, R, Popolo, L., VAI, MARINA, ALBERGHINA, LILIA |
| Přispěvatelé: |
Pignatelli, R, Vai, M, Alberghina, L, Popolo, L |
| Jazyk: |
angličtina |
| Rok vydání: |
1998 |
| Předmět: |
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| Popis: |
New secretary signals and strategies can be attempted to improve the secretion of heterologous proteins of biotechnological interest which encounter difficulties being exported in yeast. The GGPI gene of Saccharomyces cerevisiae cedes for a 125 kDa glycoprotein transported through the secretary pathway and anchored to the plasma membrane by means of a glycosylphosphatidylinositol. The regions coding for the secretary signal or also for the first 46 amino acids were tested for efficiency in secretion by fusion to the lacZ gene of Escherichia call resulting in the synthesis of the endoplasmic reticulum-targeted I-22- and I-68-GgpIp/β-gal hybrids. A cytoplasmic form was also examined. The I-22βgal is partially transported to the cell surface and in the medium in an unglycosylated form. The I-68βgal is completely retained in the intracellular membranes and is N-glycosylated in the GgpIp 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 these producing the cytosolic β-galactosidase or overexpressing an endogenous secretary protein. Thus, glycosylation and abnormal folding rather than overexpression are among the factors responsible for the decreased activity and exit of β-galactosidase from the endoplasmic reticulum and for induction of BiP. The results obtained indicate that the sole secretary signal of GgpIp 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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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