Cloning and Characterization of Carbonyl Reducing Enzymes

Members in project: Eva Novotná, Hana Štambergová, Tereza Lundová

Our cloning branch successfully established cloning of target cDNA coding sequences into different vectors for recombinant procaryotic (E. coli) and eukaryotic (insect cells, Spodoptera frugiperda Sf9) expression. Expression in E. coli is used for production of high amounts of human cytosolic enzymes from both AKR and SDR superfamily, e.g. AKR1B10, AKR1C3, CBR1, CBR3, and several mycobacterial enzymes, e.g. MenB, IcL1. Second approach is based on baculovirus expression vector system (BEVS) and it is used exclusively for production of human membrane bound enzymes from SDR group, e.g. DHRS7, DHRS3.

Main goal of this research branch is purification and liposomal reconstitution of yet unknown, membrane-bound enzymes and their detailed characterization , including localization studies, identification of substrates or screening for activity of modulators.  Moreover our group is working on already described, cytosolic carbonyl reducing enzymes, where we focus on further substrate screening and characterization, inhibition studies and the role of the enzyme inhibition in enzyme activity and its effects on metabolism.