PMID:8054847
| Citation |
Kealey, JT and Santi, DV (1994) High-level expression and rapid purification of tRNA (m5U54)-methyltransferase. Protein Expr. Purif. 5:149-52 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/prep.1994.1023 |
| Keywords |
Bacterial Proteins/biosynthesis; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Cellulose/analogs & derivatives; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; Cloning, Molecular; Enzyme Induction; Escherichia coli/enzymology; Escherichia coli/genetics; Genetic Vectors; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/isolation & purification; tRNA Methyltransferases/biosynthesis; tRNA Methyltransferases/genetics; tRNA Methyltransferases/isolation & purification |
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