Purification of Histidine- Tagged T4 RNA Ligase from E. coli
ثبت نشده
چکیده
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.
منابع مشابه
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 simpl...
متن کاملRNA nicking activity associated with DNA ligase of T4 infected E. coli: properties and influence on in vitro reactions of ligase.
Highly purified DNA ligase from T4 infected E. coli displays an RNA nicking activity which cleaves endonucleolytically the RNA of ribo-desoxy-and ribo-ribo type doublestranded structures to oligonucleotides with 5'phosphoryl-and 3'hydroxy termini. In the presence of ATP the generated nicks are repaired by the ligase except at the ends of the doublestranded regions where some short oligonucleoti...
متن کاملGenetic and physiological studies of an Escherichia coli locus that restricts polynucleotide kinase- and RNA ligase-deficient mutants of bacteriophage T4.
The RNA ligase and polynucleotide kinase of bacteriophage T4 are nonessential enzymes in most laboratory Escherichia coli strains. However, T4 mutants which do not induce the enzymes are severely restricted in E. coli CTr5X, a strain derived from a clinical E. coli isolate. We have mapped the restricting locus in E. coli CTr5X and have transduced it into other E. coli strains. The restrictive l...
متن کاملPurification and properties of a T4 bacteriophage factor that modifies valyl-tRNA synthetase of Escherichia coli.
After T4 bacteriophage infects Escherichia coli, a peptide tau, produced under the control of a phage gene, binds to the host valyl transfer ribonucleic acid synthetase (EC 6.1.1.9) and thereby changes several of its physicochemical properties. The interaction of tau with the host enzyme was investigated in vitro after extensively purifying the factor from T4-infected E. coli using a rapid puri...
متن کاملCovalent attachment of polyribonucleotides to polydeoxyribonucleotides catalyzed by deoxyribonucleic acid ligase.
DNA ligase isolated from Escherichia coli or phage T4-infected E. coli catalyzes the covalent joining of the 5’-phosphate termini of polydeoxyribonucleotides and 3’-hydroxyl termini of polyribonucleotides. This reaction occurs with poly(dA) and poly(A) in the presence of poly(dT). The 5’-phosphate terminus of poly[d(A-T)] can be linked by an intramolecular reaction to its 3’-hydroxyl terminus s...
متن کامل