Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.

中文翻译：

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使用杆状病毒系统表达和纯化重组小鼠CRISP4。

富含半胱氨酸的分泌蛋白4（CRISP4）是CAP超家族蛋白的成员，在雄性生殖道中高度表达，是哺乳动物最佳繁殖力所必需的。CRISPs的特征在于存在16个保守的半胱氨酸残基，其形成跨越N-末端CAP结构域，铰链区和C-末端离子通道调节（ICR）结构域的8个二硫键。先前纯化重组CRISPs作为一组的尝试已导致错误折叠和/或不溶的重组蛋白，蛋白聚集体或无法使用的低蛋白产率。因此，妨碍了CRISP的功能定义。在这项研究中，我们报告了使用杆状病毒表达系统在High Five™细胞中表达和纯化小鼠CRISP4蛋白的三步纯化方案。重组小鼠CRISP4通过Western印迹得到识别，并使用圆二色性（CD）在结构上进行了表征。使用本文所述的协议，我们产生了高产量的可溶性和正确折叠的重组小鼠CRISP4。