High-level expression and rapid purification of tRNA (m5U54)-methyltransferase.
We report an extremely high-level expression system for tRNA (m5U54)-methyltransferase (RUMT), and a purification strategy which routinely yields 20 to 50 mg of homogeneous RUMT per liter of Escherichia coli cells. The RUMT gene (trmA) was cloned into a pET vector and transformed into E. coli BL21 (DE3) cells. Following induction, this system produces active enzyme at a level approaching 50% of the total soluble protein. A purification scheme consisting of DEAE-cellulose chromatography to remove nucleic acids, followed by phosphocellulose chromatography, provides homogeneous enzyme. The entire procedure, from cell growth to purified enzyme, takes less than 2 days. This represents a significant 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 liter of cells and is substantially more labor intensive.
Kealey
J T
JT
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
Santi
D V
DV
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012359:tRNA Methyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C008881:phosphocellulose; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase