Study of three proteins in uracil catabolism pathway

Sammanfattning: This study focused on expression, purification and functional characterization of three key proteins (Urc1p, Urc2p and Urc8p) in the newly discovered uracil catabolism (URC) pathway. A novel expression system was established by using Saccharomyces kluyveri as a host organism for expression of 6-His tagged Urc1p and Urc2p. Moreover, URC8 gene was cloned in pET151/D-TOPO vector and expressed in Escherichia coli. All three recombined proteins were purified on nickel columns. The SDS-PAGE showed Urc1p and Urc8p are of correct size and high purity. This novel expression system provides an alternative or a complementary choice for eukaryotic protein production. The functional characterization assay suggested that Urc1p might open the pyrimidine ring of uridine in the URC pathway and Urc8p is a NADPH dependent reductase converting malonic semialdehyde to 3-hydroxypropionate, which is one of the final products in the URC pathway.