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Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate
1Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul.
Abstract:
Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.
Insights
This study expresses mouse tumor necrosis factor (TNF) precursor in yeast, finding it localizes to the cell membrane. These engineered yeast cells offer a novel system for studying TNF processing mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Tumor necrosis factor (TNF) is a key cytokine involved in inflammatory responses.
- TNF is initially produced as a membrane-bound precursor that requires cleavage for maturation.
- Understanding TNF precursor processing is crucial for developing therapies for inflammatory diseases.
Purpose of the Study:
- To express and characterize murine pro-TNF in Saccharomyces cerevisiae.
- To investigate the cellular localization and processing of recombinant pro-TNF in yeast.
- To assess the utility of engineered yeast as a model system for studying pro-TNF biology.
Main Methods:
- Gene expression of murine pro-TNF in yeast using the alcohol dehydrogenase promoter.
- Immunoblotting to detect pro-TNF expression and size.
- Cell fractionation and centrifugation to determine protein localization.
- Flow cytometry to analyze cell surface protein expression.
Main Results:
- Successfully expressed 26 kD pro-TNF in yeast, primarily localized to the cell membrane.
- Detected mature 17 kD TNF in the culture medium, though in small quantities.
- Confirmed cell surface presence of TNF via flow cytometry.
- Demonstrated membrane localization and orientation similar to mammalian cells.
Conclusions:
- Yeast can be engineered to express and correctly localize pro-TNF.
- Transformed yeast cells provide a valuable platform for genetic analysis of pro-TNF processing.
- This system facilitates research into the molecular machinery involved in TNF maturation.