A cloning and high-expression system for tRNA (m5U54)-methyltransferase (RUMT) is described. Polymerase chain reaction (PCR) was used to replicate the coding sequence and create flanking restriction sites for cloning. The PCR product was then inserted into expression vectors containing the tac and PL promoters. With the PL promoter, induced cells produced about 1.5% of their soluble protein as catalytically active RUMT. With the tac promoter, up to 8% of the total cell protein was active enzyme, and RUMT was purified to near homogeneity in three steps.