Expression, purification and crystallization of the C-terminal domain of human ubiquitin specific protease 7.

摘要

USP7 is an ubiquitin specific protease. It functions as a deubiquitinating enzyme that removes ubiquitin from target proteins. USP7 regulates the protein levels of p53, Mdm2 and MdmX. It also interacts with FOXO4 and viral proteins EBNA1, ICP0. USP7 has four domains. Two C-terminal domains which interact with ICP0 and FOXO4 are believed important for the substrate recognition, regulation and protein-protein interaction.GST pull down assays were performed to study the interaction between USP7 and ICP0. Three ICP0 peptides showed positive binding with USP7. The peptides share a 7 amino acid sequence (-RKCARKT-). ICP0 615-629 (-PRGP-RKCARKT-RHAE-) showed strongest binding ability. Four positively charged amino acids were found in the seven residues and this may suggest that these amino acids playing a role in the interactionA total of 15 C-terminal protein constructs were studied. They were prepared, expressed and purified for protein crystallization trials in order to determine their three dimensional structures. Protein precipitation, aggregation and degradation problems persisted with the nine soluble proteins and these are negative factors for protein crystallization. Four proteins have been crystallized and more work must be done to improve the crystals.