Prokaryotic expression, purification, and functional characterization of recombinant buffalo (Bubalus bubalis) cysteine-rich secretory protein 1 on sperm physiology
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View abstract on PubMed
Summary
This summary is machine-generated.Recombinant buffalo Cysteine-rich secretory protein 1 (CRISP-1) was produced in E. coli. This bioactive protein inhibits sperm motility and capacitation, showing potential for buffalo semen preservation.
Area Of Science
- Reproductive Biology
- Biochemistry
- Biotechnology
Background
- Cysteine-rich secretory protein 1 (CRISP-1) is an epididymis glycoprotein known to decapacitate sperm.
- Preventing premature sperm capacitation is crucial for extending fertility during semen preservation.
Purpose Of The Study
- To produce bioactive recombinant buffalo CRISP-1 in Escherichia coli.
- To evaluate its effects on buffalo sperm motility, capacitation, and signaling pathways.
Main Methods
- Optimized expression and purification of buffalo CRISP-1 (C15-C242) using E. coli strains and Ni-NTA affinity chromatography.
- Confirmed protein identity via mass spectrometry.
- Assessed functional activity across different pH and temperatures.
Main Results
- Successfully produced and purified (>90%) bioactive recombinant buffalo CRISP-1.
- Significantly reduced sperm progressive motility and capacitation, inhibiting key protein tyrosine phosphorylation (p47, p72).
- Demonstrated activity between pH 6.0-9.0 (optimum pH 8.0), with thermolability above 60°C. Inhibited HCO₃⁻ and L-arginine-induced capacitation via NO*, AC/cAMP, and Ca²⁺ pathways.
Conclusions
- Recombinant buffalo CRISP-1 is bioactive and effectively inhibits sperm motility and capacitation.
- CRISP-1 shows promise for application in buffalo semen preservation to mitigate cryocapacitation.
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