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Biochemical characterization of zebrafish Prss59.1
Rubel Rana1, Forhad Hossain2, Hasan Ali1
1Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
Summary
Zebrafish prss59.1, a candidate ovulation-inducing gene, was biochemically characterized. Recombinant Prss59.1 showed trypsin-like peptidase activity, with optimal conditions at 37°C and pH 8.0.
Area of Science:
- Reproductive biology
- Molecular genetics
- Biochemistry
Background:
- prss59.1 identified as a candidate ovulation-inducing gene via transcriptomic analysis.
- Ongoing development of a prss59.1 gene knock-out zebrafish model for functional studies.
Purpose of the Study:
- To biochemically characterize zebrafish Prss59.1 (a putative ovulation-inducing gene).
- To determine the enzymatic activity and optimal conditions for Prss59.1.
Main Methods:
- Construction and expression of a C-terminal histidine-tagged recombinant zebrafish Prss59.1 protein in E. coli.
- Biochemical assays to assess peptidase activity, optimal temperature, pH, and substrate specificity (Lys-MCA).
- Denaturation and renaturation steps were employed to induce protein activity.
Main Results:
- Recombinant Prss59.1 exhibited minimal activity in its native state but showed significant peptidase activity after denaturation and renaturation.
- The enzyme displayed highest activity against Lys-MCA.
- Optimal activity was observed at 37°C and pH 8.0, with a Km value of 0.17 mM.
Conclusions:
- Zebrafish Prss59.1 possesses trypsin-like peptidase characteristics, consistent with its DNA sequence.
- These findings provide foundational biochemical data for understanding the role of prss59.1 in zebrafish reproduction.
