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A signal sequence detection system using secreted protease activity as an indicator
M Yokoyama-Kobayashi1, S Sugano, T Kato
1Kanagawa Academy of Science and Technology (KAST), Japan.
Gene
|October 3, 1995
Summary
We created a new tool, pSSD1, to identify signal sequences (Ss) in secreted proteins. This method detects intact Ss by measuring fibrinolytic activity, aiding in the discovery of secreted and membrane proteins.
Area of Science:
- Molecular Biology
- Protein Secretion
- Gene Expression Vectors
Background:
- Secretory signal sequences (Ss) are crucial for protein targeting to secretion pathways.
- Efficiently identifying cDNAs encoding secreted or membrane proteins is essential in molecular biology.
Purpose of the Study:
- To develop a novel expression vector, pSSD1, for detecting cDNA fragments encoding functional secretory signal sequences (Ss).
- To validate the utility of pSSD1 in identifying secreted proteins through a reporter gene assay.
Main Methods:
- Construction of the pSSD1 expression vector containing a human urokinase-type plasminogen activator (u-PA) protease domain reporter gene.
- Insertion of various cDNA fragments encoding putative Ss upstream of the u-PA reporter gene.
- Transfection of monkey COS7 cells with engineered plasmids and assessment of secreted fibrinolytic activity.
Main Results:
- Fibrinolytic activity was detected in the culture medium only when intact secretory signal sequences were present.
- The pSSD1 vector successfully identified functional Ss, demonstrating its efficacy.
- Chimeric proteins were produced in transfected cells, with activity correlating to Ss integrity.
Conclusions:
- The pSSD1 vector provides a reliable system for detecting cDNA fragments encoding functional secretory signal sequences.
- This approach is valuable for identifying cDNAs of secreted proteins and type-I membrane proteins.
- The developed system facilitates research in protein trafficking and secretion pathways.