High-level expression of Escherichia coli tRNA (m5U54)-methyltransferase.
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.
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Department of Biochemistry, University of California, San Francisco 94143.
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D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012359:tRNA Methyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase