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cDNA cloning and sequence analysis of a novel calcium binding protein with oligoproline motif
1Department of Biochemistry, Faculty of Medicine, Kagoshima University, Japan.
Abstract:
Using antibodies against pokeweed agglutinin-binding proteins from F9 embryonal carcinoma cells, we isolated a cDNA clone PW29 reacting with the antibody. mRNA hybridizing with the cDNA was 4.7 kb, and was strongly expressed in the testis, brain, kidney and heart as well as in F9 cells. The size of mRNA in the testis was heterogeneous. Sequencing the cDNA clone revealed a putative polypeptide of 70 kDa, which is rich in hydrophilic amino acids and has a characteristic sequence of (Glu(Lys))5. The protein also has an oligoproline motif, which conforms to the rule of binding capability to Src homology region III. The cDNA was translated as a fusion protein with glutathione-S-transferase, and was verified to have calcium binding activity, upon a calcium blot experiment.
Insights
Researchers identified a novel protein, PW29, from embryonal carcinoma cells. This protein, expressed in various tissues, exhibits calcium-binding activity and may interact with Src homology region III.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- F9 embryonal carcinoma cells express pokeweed agglutinin-binding proteins.
- Antibodies were generated against these proteins to identify related cDNA clones.
Purpose of the Study:
- To isolate and characterize a novel cDNA clone, PW29, from F9 cells.
- To investigate the expression patterns and functional properties of the protein encoded by PW29.
Main Methods:
- Antibody screening of a cDNA library from F9 cells.
- Northern blot analysis to determine mRNA expression.
- DNA sequencing to predict polypeptide structure.
- In vitro translation and calcium blot assay to assess protein function.
Main Results:
- A cDNA clone, PW29, was isolated, with its mRNA (4.7 kb) strongly expressed in testis, brain, kidney, heart, and F9 cells.
- The predicted 70 kDa polypeptide is hydrophilic, contains a (Glu(Lys))5 repeat, and an oligoproline motif.
- The protein demonstrated calcium-binding activity.
Conclusions:
- PW29 represents a novel protein with potential roles in various tissues, including reproductive and neural systems.
- The identified protein possesses calcium-binding capabilities and structural motifs suggesting interactions with other cellular components.