Prokaryotic expression, purification, and functional characterization of recombinant buffalo (Bubalus bubalis)...

Cysteine-rich secretory protein 1 (CRISP-1), an epididymis-origin glycoprotein, is recognized as a sperm decapacitation factor. Incorporation of CRISP-1 in semen prior to preservation may extend sperm fertility by preventing premature capacitation. This study aimed to produce bioactive recombinant buffalo CRISP-1 in Escherichia coli and evaluate its effects on sperm motility, capacitation, and associated signalling mechanisms. The expression of a 27.6 kDa fragment of buffalo CRISP-1 (C15-C242) protein was optimized by using E. coli BL21(DE3) and BL21(DE3)-CodonPlus strains under different IPTG concentrations and induction temperatures. Purification by Ni-NTA affinity chromatography was optimized by employing various lysis and washing buffers, resulting >90 % purity and modest yield. The identity of the protein was confirmed by mass spectrometry. Functionally, the protein significantly reduced sperm progressive motility (from 61 % to 33 %) and capacitation. It also inhibited tyrosine phosphorylation of key sperm proteins (p47 and p72) under capacitating conditions. The protein retained activity across pH 6.0-9.0 (optimum at pH 8.0) but was thermolabile above 60 °C. Expression of CRISP-1 was found in the vas deferens, corpus, and cauda epididymis, but not in the testes. In sperm, it is localized both in the acrosome and the flagellum. Mechanistically, CRISP-1 inhibited both HCO₃^- and L-arginine-induced capacitation by modulating nitric oxide (NO*), adenylyl cyclase (AC)/cAMP, and calcium (Ca²⁺) signalling pathways. In conclusion, recombinant buffalo CRISP-1 was successfully produced in E. coli and shown to be bioactive. It effectively inhibited sperm motility and capacitation, indicating its possible application in semen preservation for reducing cryocapacitation in buffalo sperm.