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 | Organism |
---|---|
protein is stable at -20°C for several months | Leishmania tarentolae |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
mitochondrion | - |
Leishmania tarentolae | 5739 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | assay with 10 mM Mg2+ | Trypanosoma brucei | |
Mg2+ | assay with 10 mM Mg2+ | Leishmania tarentolae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Leishmania tarentolae | - |
- |
- |
Trypanosoma brucei | - |
- |
- |
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 | Organism |
---|---|
protein remains stable at -20°C in 50% glycerol up to 3 months | Trypanosoma brucei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
UTP + RNAn | - |
Trypanosoma brucei | diphosphate + RNAn+1 | - |
? | |
UTP + RNAn | - |
Leishmania tarentolae | diphosphate + RNAn+1 | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | - |
Leishmania tarentolae |
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 Maximum [°C] | Comment | Organism |
---|---|---|---|
27 | - |
assay at | Trypanosoma brucei |
27 | - |
assay at | Leishmania tarentolae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Trypanosoma brucei |
8 | - |
assay at | Leishmania tarentolae |