Expression and purification of recombinant mouse CRISP4 using a baculovirus system
Expression and purification of recombinant mouse CRISP4 using a baculovirus system
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DOI：
10.1016/j.pep.2019.105543
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发表时间：
2020-03-01
影响因子：
1.6
通讯作者：
O'Bryan, Moira K.
中科院分区：
文献类型：
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作者：
Gaikwad, Avinash S.;Loh, Khai Lee;O'Bryan, Moira K.
富含半胱氨酸分泌蛋白4 （CRISP4）是CAP超家族蛋白的一员，在雄性生殖道中高度表达，是哺乳动物最佳生育能力所必需的。CRISPs的特点是存在16个保守的半胱氨酸残基，形成8个二硫键，分布在n端CAP结构域、铰链区域和c端离子通道调节（ICR）结构域。先前纯化重组CRISPs的尝试导致错误折叠和/或不溶性重组蛋白，蛋白质聚集或无法使用的低蛋白产量。因此，定义CRISPs的功能受到了阻碍。在这项研究中，我们报道了一种利用杆状病毒表达系统在High Five （TM）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 blotting进行识别，并利用圆二色性（Circular Dichroism， CD）进行结构表征。使用本文描述的方案，我们产生了高产量的可溶性和正确折叠的重组小鼠CRISP4。
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 (TM) 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.