利用桿狀病毒系統表現SUMOylated Human DNA Topoisomerase Ι

Abstract

SUMOylation is a post-translational modification involved in various cellular processes. SUMO (small ubiquitin-related modifier) protein conjugates to lysine residue covalently and further modify the functions of its target proteins. Human topoisomerase I (hTOP1) is an enzyme that acts on the topology of DNA during cell replication and transcription, as well as during chromosome condensation and segregation. Previous work done in our laboratory had shown that hTOP1 in cells treated with a TOP1 inhibitor, camptothecin, is vulnerable to SUMO1, but the mechanism associated with this phenomena is unclear. In this study, we attempt to express the SUMOylated hTOP1. It has been demonstrated that the level of SUMOylated protein in mammalian expression system is less than 5% of the total protein levels of a cell, and this will inevitably cause difficulties for purification of the SUMOylated protein. To date, the baculovirus expression system is one of the powerful tools employed for large scale protein production. In our study, separated recombinant baculovirus vector containing genes that encode components of SUMOylation (E1, hUbc9, SUMO1) and hTOP1 were used to infect Sf21 insect cell line. Our aim is to express large quantity of the SUMOylated-hTOP1 protein in the Sf21 insect cells in order to facilitate purification of the SUMOylated-hTOP1 protein for further analysis. As shown in this work, the SUMOylated-hTOP1 protein can be highly expressed by the baculovirus vector system thus providing an alternative way for large-scale expression of SUMOylated protein.