Centro de Investigación Príncipe Felipe, Valencia, Spain.
Protein J. 2010 Feb;29(2):69-74. doi: 10.1007/s10930-009-9223-y.
MCT-1 protein is encoded by an oncogene highly expressed in lymphomas. It is implicated in the interaction with the cap complex of the mRNA, through an RNA binding domain, named PUA. Targeted suppression of this domain attenuates the malignant phenotype and hence MCT-1 is a potential target for therapeutic intervention. In the present study 6 xHis-tagged MCT-1 expression and purification was assessed in insect cells using a baculovirus expression system. The gene was amplified by PCR from a human cDNA library, encoding an open reading frame of 181 amino acid residues. High MCT-1 production level (6 mg/L) was achieved in a two-step purification procedure. The protein was partially characterized by gel filtration chromatography, peptide mass fingerprinting and circular dichroism. A cap-binding assay confirmed its appropriate folding and functionality. Furthermore, a three dimensional model was built based on another known PUA domain structure. The abundant, pure and properly folded source of MCT-1 protein generated lays a foundation for future structure-function studies.
MCT-1 蛋白由淋巴瘤中高表达的癌基因编码。它通过一个名为 PUA 的 RNA 结合结构域与 mRNA 的帽复合物相互作用。靶向抑制该结构域可减弱恶性表型，因此 MCT-1 是治疗干预的潜在靶点。在本研究中，使用杆状病毒表达系统在昆虫细胞中评估了 6xHis 标记的 MCT-1 表达和纯化。该基因通过从人类 cDNA 文库扩增的 PCR 扩增，编码 181 个氨基酸残基的开放阅读框。通过两步纯化程序实现了高 MCT-1 生产水平（6mg/L）。通过凝胶过滤层析、肽质量指纹图谱和圆二色性对蛋白质进行了部分表征。帽结合测定证实了其适当的折叠和功能。此外，还基于另一个已知的 PUA 结构域构建了一个三维模型。生成的丰富、纯净和正确折叠的 MCT-1 蛋白源为未来的结构-功能研究奠定了基础。
