HIGH-LEVEL EXPRESSION AND RAPID PURIFICATION OF TRANSFER-RNA (M(5)U54)-METHYLTRANSFERASE

We report an extremely high-level expression system for tRNA (m(5)U54) -methyltransferase (RUMT), and a purification strategy which routinely yields 20 to 50 mg of homogeneous RUMT per liter of Escherichia coil cells. The RUMT gene (trmA) was cloned into a pET vector and transform ed into E. coil BL21 (DE3) cells. Following induction, this system pro duces active enzyme at a level approaching 50% of the total soluble pr otein. A purification scheme consisting of DEAE-cellulose chromatograp hy to remove nucleic acids, followed by phosphocellulose chromatograph y, provides homogeneous enzyme. The entire procedure, from cell growth to purified enzyme, takes less than 2 days. This represents a signifi cant improvement over the previously published expression/purification protocol for RUMT (Gu, X, and Santi, D. V., Protein Expression Purif. 2, 66-68, 1991), which typically nets 5- to 10-fold less enzyme per l iter of cells and is substantially more labor intensive. (C) 1994 Acad emic Press, Inc.