Department of Veterinary Biochemistry, Rajasthan University of Animal and Veterinary Sciences, Bikaner, India.
Division of Animal Physiology & Biochemistry, ICAR-Central Sheep and Wool Research Institute, Avikanagar, Jaipur, India.
Syst Biol Reprod Med. 2021 Dec;67(6):471-481. doi: 10.1080/19396368.2021.1963012. Epub 2021 Aug 29.
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 C peptide fragment of ovine CRISP-1 protein was cloned into THE pET32b(+) expression vector using DH5α. Protein expression was carried out in 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 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. CRISP-1: Cysteine-rich secretory protein-1;
绵羊半胱氨酸丰富的分泌蛋白 1（CRISP-1）是一种酸性糖蛋白，来源于附睾，属于 CRISP、抗原 5、与疾病相关的蛋白 1（CAP）超家族。本研究的目的是优化细菌生产并对推测成熟的绵羊 CRISP-1 蛋白进行部分特性分析。使用 DH5α 将与绵羊 CRISP-1 蛋白的 T C 肽片段对应的 cDNA 克隆到 pET32b(+)表达载体中。在 BL21(DE3)中通过 1mM IPTG 在 37°C 诱导 4 小时进行蛋白表达。重组蛋白作为包涵体表达，并通过 Ni-NTA 亲和层析使用 pH 梯度进行纯化。进一步通过锌离子负染色后的凝胶提取对蛋白进行纯化。纯化的重组 CRISP-1 蛋白的 SDS-PAGE 分析显示出 43.8 kDa 的条带。对纯化的 CRISP-1 蛋白的生物活性进行了精子活力和获能的检测。5μg/ml 的重组绵羊 CRISP-1 蛋白显著抑制精子活力，并且将蛋白在 100°C 加热 5 分钟后活性丧失。该蛋白还表现出脱帽活性，在 2μg/ml 的浓度下，显著（P<0.05）降低精子获能。总之，成功地在 BL21(DE3)中产生了硫氧还蛋白标记的绵羊 CRISP-1 蛋白，并通过 Ni-NTA 亲和层析、凝胶纯化和透析的组合以可溶性形式进行了纯化。重组蛋白表现出抑制活力和脱帽活性。需要进一步研究以阐明其作用机制，并评估其在精液保存中的潜在用途。CRISP-1：富含半胱氨酸的分泌蛋白-1。
