Hopson Sarah E, Thompson Martin J
Department of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI, 49931, United States of America.
Department of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI, 49931, United States of America.
Protein Expr Purif. 2018 Dec;152:23-30. doi: 10.1016/j.pep.2018.07.002. Epub 2018 Jul 18.
The human polybromo-1 protein (BAF180) is a known driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. BAF180 is the chromatin-targeting subunit of the PBAF complex. BAF180 has six bromodomains, two BAH domains, and one HMG box. Bromodomains are known to recognize acetylated-lysines on histones and play a role in nucleosome recognition. BAH domains are required for ubiquitination of PCNA, a key regulator of DNA damage. The putative HMG box, if functional, may be involved in DNA-binding. While the binding specificities of individual bromodomains have been studied by our lab and others, the results have failed to reach a consensus. The acetyl-histone binding features of the full-length protein is unknown and is the motivation for this work. The hypothetical HMG and BAH domains have not been studied and the actual function of these regions is currently unknown. Thus, the precise interactions of this large and complex protein are not well-studied. Advances in understanding this large protein have been hindered by the inability to express and purify recombinant full-length BAF180 protein. Currently, only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. Here, we report the successful expression, purification of full-length biologically active BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris. The ability to express full-length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression.
人类多溴-1蛋白（BAF180）是透明细胞肾细胞癌中一种已知的驱动突变，约40%的病例中存在该蛋白的突变。BAF180是PBAF复合物的染色质靶向亚基。BAF180有六个溴结构域、两个BAH结构域和一个HMG框。已知溴结构域可识别组蛋白上的乙酰化赖氨酸，并在核小体识别中发挥作用。BAH结构域是PCNA泛素化所必需的，PCNA是DNA损伤的关键调节因子。假定的HMG框若具有功能，可能参与DNA结合。虽然我们实验室和其他研究团队已对单个溴结构域的结合特异性进行了研究，但结果尚未达成共识。全长蛋白与乙酰化组蛋白的结合特性尚不清楚，这也是开展本研究的动机。假设的HMG和BAH结构域尚未得到研究，这些区域的实际功能目前也不清楚。因此，这种大而复杂的蛋白的精确相互作用尚未得到充分研究。由于无法表达和纯化重组全长BAF180蛋白，对这种大蛋白的理解进展受到了阻碍。目前，仅进行了使用在哺乳动物细胞中表达的BAF180的现象学研究。在此，我们报告了利用异源宿主巴斯德毕赤酵母中的GAP启动子成功表达、纯化出全长具有生物活性的BAF180蛋白。表达全长和突变型BAF180的能力将有助于进行生物物理结合研究。了解结合相互作用对于我们理解BAF180在癌症发生发展中的作用至关重要。
