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

• Published in: PloS one 2023-02, Vol.18 (2), p.e0277478-e0277478
• Main Authors: Russell, Bonnie L, Ntwasa, Monde
• Format: Article
• Language: English
• Subjects: Analysis; Antibodies; Biology and Life Sciences; Biomarkers; Cancer; Carcinogenesis; Carcinogens; Carrier Proteins - metabolism; Cell cycle; Chromatography; Computer and Information Sciences; Diagnosis; DNA-Binding Proteins - metabolism; E coli; Embryogenesis; Embryonic development; Embryonic growth stage; Engineering and Technology; Genetic aspects; Health aspects; Helices; Mass spectrometry; p53 Protein; Physical Sciences; Polyhistidine; Protein Binding; Protein purification; Protein structure; Proteins; Purification; Random coil; Research and Analysis Methods; Retinoblastoma; Retinoblastoma Binding Proteins; Scientific imaging; Secondary structure; Sodium; Therapeutic targets; Tumor proteins; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; Ubiquitin-Protein Ligases - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0277478

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.