Development of a Recombinant Protein Expression System for the Production of Human Carbonyl Reductase
Dr. Henry Charlier and Dr. Kristen Mitchell
Anthracyclines are commonly used to treat a variety of cancers, but are limited in use by a dose-dependent cardiotoxicity. This cardiotoxicity is linked to the enzymatic reduction of the anthracyclines by human carbonyl reductase (HCBR). Important to the ongoing studies is the development of a readily available source of human carbonyl enzyme. Recombinant forms of HCBR were developed whereby peptides harboring a 6X-polyhistidine tail were added to either the amino- or carboxy-terminus of the protein for purification via nickel affinity chromatography. The protein with the carboxy-terminal modification had markedly reduced turnover number and catalytic efficiency. The specific activity of the amino-terminal modified enzyme is closer to the native enzyme. Preliminary results indicate HCBR is less affected by amino-terminal modification than modification at the carboxy-terminus (NIH Grant # P20 RR016454).