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Literature summary for 2.7.7.52 extracted from
- RNA editing uridylyltransferases of trypanosomatids (2007), Methods Enzymol., 424, 55-73.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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 |
| 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 |
| 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 |
| TbRET2 fused with the affinity TAP tag at the C-terminus using pLew79 vector in 29-13 cell line | Trypanosoma brucei |
General Stability
| General Stability | Organism |
|---|---|
| protein is stable at -20°C for several months | Leishmania tarentolae |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| mitochondrion | - |
Leishmania tarentolae | 5739 | - |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | assay with 10 mM Mg2+ | Trypanosoma brucei |  |
| Mg2+ | assay with 10 mM Mg2+ | Leishmania tarentolae | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Leishmania tarentolae | - |
- |
- |
| Trypanosoma brucei | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| expression and purification of 20S editosome-associated RET2 complex from Trypanosoma brucei by affinity isolation | Trypanosoma brucei |
| 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 |
| 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 |
| 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 |
| 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
| Storage Stability | Organism |
|---|---|
| protein remains stable at -20°C in 50% glycerol up to 3 months | Trypanosoma brucei |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| UTP + RNAn | - |
Trypanosoma brucei | diphosphate + RNAn+1 | - |
? | |
| UTP + RNAn | - |
Leishmania tarentolae | diphosphate + RNAn+1 | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homotetramer | - |
Leishmania tarentolae |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| RET1 | - |
Trypanosoma brucei |
| RET1 | - |
Leishmania tarentolae |
| RET2 | - |
Trypanosoma brucei |
| terminal RNA uridylyltransferase | - |
Trypanosoma brucei |
| terminal RNA uridylyltransferase | - |
Leishmania tarentolae |
| TUTase | - |
Trypanosoma brucei |
| TUTase | - |
Leishmania tarentolae |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 27 | - |
assay at | Trypanosoma brucei |
| 27 | - |
assay at | Leishmania tarentolae |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Trypanosoma brucei |
| 8 | - |
assay at | Leishmania tarentolae |
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Release 2023.1 (January 2023)
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