Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate
Experimental & Molecular Medicine
;
: 61-66, 2000.
Article
in English
| WPRIM
| ID: wpr-75103
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Protein Precursors
/
Saccharomyces cerevisiae
/
Transformation, Genetic
/
Immunoblotting
/
Cell Line
/
Cell Membrane
/
Tumor Necrosis Factor-alpha
/
Flow Cytometry
/
Animals
Limits:
Animals
Language:
English
Journal:
Experimental & Molecular Medicine
Year:
2000
Type:
Article
Similar
MEDLINE
...
LILACS
LIS