Purification of histidine-tagged T4 RNA ligase from E. coli.
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.
Wang
Qing S
QS
Simon Fraser University, 8888 University Dr., Burnaby, B.C., V5A 1S6, Canada.
Unrau
Peter J
PJ
D017122:Bacteriophage T4 / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000032:analysis*; D008969:Molecular Sequence Data; D009532:Nickel; D009571:Nitrilotriacetic Acid; D011130:RNA Ligase (ATP) / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C033223:polyhistidine; D006639:Histidine; D009532:Nickel; D012685:Sepharose; D011130:RNA Ligase (ATP); C467724:bacteriophage T4 RNA ligase 2; D009571:Nitrilotriacetic Acid