Production of recombinant human proliferating cellular nuclear antigen (PCNA) for structural and biophysical characterization.

Human proliferating cell nuclear antigen (hPCNA) is a DNA replication processivity factor, which acts as a docking platform, allowing proteins to have access to the replication fork and increasing the affinity of DNA interacting proteins, making it critical for cell survival. The trimer forms a ring-shaped oligomer allowing DNA to pass through the middle and interacting proteins to dock on the outside of the ring. Without this structural formation, there is a loss of DNA replication and repair in the cell. Due to the location of subunit-subunit termini, the addition of a purification tag can hamper crystallography and biophysical experiments, as the trimer complex folding can be impeded. To avoid these complications, a tag-less, step-wise purification was implemented, which resulted in 17.6 mg from 2 L culture of pure hPCNA with a 260 nm/280 nm value of 0.43. The produced crystal structure reveals a correctly formed oligomer. The clear depletion of the tracer binding and probe protein interaction in a fluorescence polarisation competition-based assay demonstrates the purification method produces a protein structure with a functional binding site. This purification method presents a reliable and simple method for producing hPCNA for biophysical characterisation.

Fractional Deletion of Compound Kushen Injection Indicates Cytokine Signaling Pathways are Critical for its Perturbation of the Cell Cycle.

We used computational and experimental biology approaches to identify candidate mechanisms of action of aTraditional Chinese Medicine, Compound Kushen Injection (CKI), in a breast cancer cell line (MDA-MB-231). Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis. The initial fractionation separated major from minor compounds, and it showed that major compounds accounted for little of the activity of CKI. Furthermore, removal of no single major compound altered the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical with respect to CKI activity. Transcriptome analysis was used to correlate compound removal with gene expression and phenotype data. Many compounds in CKI are required to trigger apoptosis but significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.