Production of bioactive recombinant ovine cysteine-rich secretory protein 1 in Escherichia coli.

Abstract

Ovine cysteine-rich secretory protein 1 (CRISP-1) is an acidic glycoprotein of epididymal origin under CRISP, antigen 5, pathogenesis-related protein 1 (CAP) super-family. The aim of the present study was the optimization of bacterial production and partial characterization of putative mature ovine CRISP-1 protein. The cDNA corresponding to T^{23 -} C²⁴² peptide fragment of ovine CRISP-1 protein was cloned into THE pET32b(+) expression vector using E. coli DH5α. Protein expression was carried out in E. coli BL21(DE3) by inducition with 1 mM IPTG at 37°C for 4 h. The recombinant protein was expressed as inclusion bodies and purified by Ni-NTA affinity chromatography using a pH gradient. Further purification of the protein was carried out by gel extraction following zinc sulfate negative staining. SDS-PAGE analysis of the purified recombinant CRISP-1 protein revealed a 43.8 kDa band. Bioactivity of the purified CRISP-1 protein was examined on sperm motility and capacitation. The recombinant ovine CRISP-1 protein at 5 µg/ml caused significant inhibition of sperm motility, and the activity was lost following heating the protein at 100°C for 5 min. The protein also demonstrated decapacitation activity, and at a concentration of 2 µg/ml, it caused a significant (P < 0.05) reduction in sperm capacitation. In conclusion, the thioredoxin-tagged ovine CRISP-1 protein was successfully produced in E. coli and purified in the soluble form by a combination of Ni-NTA affinity chromatography, gel purification, and dialysis. The recombinant protein exhibited both motility-inhibiting and decapacitating activities. Further study is needed to elucidate the mechanism of action and evaluate it's possible use in semen preservation.Abbreviations: CRISP-1: Cysteine-rich secretory protein-1; PCR: polymerase chain reaction; IPTG: isopropyl-β-D-thiogalactopyranoside; LB: Luria Bertani; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA: ethylene diamine tetraacetic acid; Ni-NTA: Nickel nitrilotriacetic acid.