Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate.
- Author:
Jae Yeon JEONG
1
;
Dae Myung JUE
Author Information
1. Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul.
- Publication Type:Research Support, Non-U.S. Gov't
- Keywords:
Tumor necrosis factor;
protein processing;
post-translational;
Saccharomyces cerevisiae;
cell surface proteins;
membrane proteins
- MeSH:
Animal;
Cell Line;
Cell Membrane/metabolism;
Flow Cytometry;
Immunoblotting;
Mice;
Plasmids;
Protein Precursors/metabolism*;
Protein Precursors/genetics;
Saccharomyces cerevisiae/metabolism*;
Saccharomyces cerevisiae/genetics;
Transformation, Genetic;
Tumor Necrosis Factor/metabolism*;
Tumor Necrosis Factor/genetics
- From:Experimental & Molecular Medicine
2000;32(2):61-66
- CountryRepublic of Korea
- Language:English
-
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.
