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Literature summary extracted from

  • Aphasizhev, R.; Aphasizheva, I.
    RNA editing uridylyltransferases of trypanosomatids (2007), Methods Enzymol., 424, 55-73.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.52 expression in Escherichia coli BL21(DE3) Codon Plus RIL, use of strains bearing additional tRNAs for expression of genes with G-rich codons is essential for RET1 expression Trypanosoma brucei
2.7.7.52 expression in Escherichia coli BL21(DE3) Codon Plus RIL, use of strains bearing additional tRNAs for expression of genes with G-rich codons is essential for RET1 expression Leishmania tarentolae
2.7.7.52 for recombinant expression in Escherichia coli BL21 the RET2 gene lacking 21 codons at the 5' end, which correspond to the mitochondria importation signal, is cloned into pET15+vector to generate N-terminal 6 His-RET2 fusion protein Trypanosoma brucei
2.7.7.52 TbRET2 fused with the affinity TAP tag at the C-terminus using pLew79 vector in 29-13 cell line Trypanosoma brucei

General Stability

EC Number General Stability Organism
2.7.7.52 protein is stable at -20°C for several months Leishmania tarentolae

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.7.7.52 mitochondrion
-
 Leishmania tarentolae 5739
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.7.52 Mg2+ assay with 10 mM Mg2+ Trypanosoma brucei
2.7.7.52 Mg2+ assay with 10 mM Mg2+ Leishmania tarentolae

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.52 Leishmania tarentolae
-
-
-
2.7.7.52 Trypanosoma brucei
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.52 expression and purification of 20S editosome-associated RET2 complex from Trypanosoma brucei by affinity isolation Trypanosoma brucei
2.7.7.52 homogenous RET1 is purified from an enriched mitochondrial fraction of Leishmania tarentolae by a series of chromatographic steps: affinity chromatography (polyU Sepharose 4B), anion-exchange chromatography (Poros 20 HQ 46/100 column), gel filtration (Superose 12 and Superose 6 columns) and hydrophobic interactions chromatography (Phenyl Superose column) Leishmania tarentolae
2.7.7.52 recombinant RET1 from Leishmania tarentolae is purified by cation-exchange resin (Sepharose S Fast Flow), metal affinity chromatography (Talon metal affinity column), anion-exchange chromatography (Mono Q 5/5 column), expected yield: 0.2-0.5 mg/liter Leishmania tarentolae
2.7.7.52 recombinant RET1 from Trypanosoma brucei is purified by metal affinity chromatography (Talon metal affinity column), anion-exchange chromatography (HiTrap Q column) and loading onto a MonoS 5/5 column (final purification step), expected yield is 0.5 mg/liter of bacterial culture, it is important to maintain the concentration of KCl above 80 mM during purification, as protein tends to precipitate under low salt conditions Trypanosoma brucei
2.7.7.52 the recombinant expressed RET2 is purified by loading the protein extract onto a Talon affinity column and onto a HiTrap Sepharose S column, the expected protein yield is 1 mg of protein per liter of bacterial culture Trypanosoma brucei

Storage Stability

EC Number Storage Stability Organism
2.7.7.52 protein remains stable at -20°C in 50% glycerol up to 3 months Trypanosoma brucei

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.52 UTP + RNAn
-
 Trypanosoma brucei diphosphate + RNAn+1
-
 ?
2.7.7.52 UTP + RNAn
-
 Leishmania tarentolae diphosphate + RNAn+1
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.7.52 homotetramer
-
 Leishmania tarentolae

Synonyms

EC Number Synonyms Comment Organism
2.7.7.52 RET1
-
 Trypanosoma brucei
2.7.7.52 RET1
-
 Leishmania tarentolae
2.7.7.52 RET2
-
 Trypanosoma brucei
2.7.7.52 terminal RNA uridylyltransferase
-
 Trypanosoma brucei
2.7.7.52 terminal RNA uridylyltransferase
-
 Leishmania tarentolae
2.7.7.52 TUTase
-
 Trypanosoma brucei
2.7.7.52 TUTase
-
 Leishmania tarentolae

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.7.52 27
-
 assay at Trypanosoma brucei
2.7.7.52 27
-
 assay at Leishmania tarentolae

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.7.52 8
-
 assay at Trypanosoma brucei
2.7.7.52 8
-
 assay at Leishmania tarentolae