Expression, purification, and characterisation of the p53 binding domain of Retinoblastoma binding protein 6 (RBBP6)

Abstract

RBBP6 is a 250 kDa eukaryotic protein known to be a negative regulator of p53 and essential for embryonic development. Furthermore, RBBP6 is a critical element in carcinogenesis and has been identified as a potential biomarker for certain cancers. RBBP6’s ability to interact with p53 and cause its degradation makes it a potential drug target in cancer therapy. Therefore, a better understating of the p53 binding domain of RBBP6 is needed. This study presents a three-part purification protocol for the polyhistidine-tagged p53 binding domain of RBBP6, expressed in Escherichia coli bacterial cells. The purified recombinant domain was shown to have structure and is functional as it could bind endogenous p53. We characterized it using clear native PAGE and far-UV CD and found that it exists in a single form, most likely monomer. We predict that its secondary structure is predominantly random coil with 19% alpha-helices, 9% beta-strand and 14% turns. When we exposed the recombinant domain to increasing temperature or known denaturants, our investigation suggested that the domain undergoes relatively small structural changes, especially with increased temperature. Moreover, we notice a high percentage recovery after returning the domain close to starting conditions. The outcome of this study is a pure, stable, and functional recombinant RBBP6-p53BD that is primarily intrinsically disordered.

Probing the Effects of Retinoblastoma Binding Protein 6 (RBBP6) Knockdown on the Sensitivity of Cisplatin in Cervical Cancer Cells

“…Accumulating studies have demonstrated that p53 is mutated in various cancers; however, most individuals with CC express wtp53 [25,26]. Recent studies have reported that p53 is negatively regulated by the retinoblastoma binding protein 6 (RBBP6) gene, a eukaryotic protein with a size of 250 kDa, responsible for embryonic development [27]. The RBBP6 gene is alternatively spliced to generate three protein isoforms that are differentially expressed in different tumor stages, with more advanced cancers exhibiting higher levels of RBBP6 compared to less advanced stages [28,29].…”