Abstract
Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.
Publication types
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Research Support, Non-U.S. Gov't
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Technical Report
MeSH terms
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Bacteriophage T4 / genetics
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Base Sequence
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Chromatography, Affinity / methods*
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Escherichia coli / chemistry*
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Escherichia coli / metabolism
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Histidine / analysis*
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Molecular Sequence Data
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Nickel
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Nitrilotriacetic Acid
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RNA Ligase (ATP) / genetics
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RNA Ligase (ATP) / isolation & purification*
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RNA Ligase (ATP) / metabolism
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Sepharose
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Viral Proteins / genetics
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Viral Proteins / isolation & purification*
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Viral Proteins / metabolism
Substances
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Recombinant Fusion Proteins
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Viral Proteins
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polyhistidine
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Histidine
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Nickel
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Sepharose
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RNA Ligase (ATP)
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bacteriophage T4 RNA ligase 2
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Nitrilotriacetic Acid