Literature summary extracted from
Lim, S.I.; Mizuta, Y.; Takasu, A.; Kim, Y.H.; Kwon, I.
Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activity (2014), PLoS ONE, 9, e98403 .
Cloned(Commentary)
EC Number |
Cloned (Comment) |
Organism |
---|
1.5.1.3 |
recombinant expression of His-tagged wild-type mDHFR and modified mDHFR-43pEthF enzymes in Escherichia coli strain XL1-Blue |
Mus musculus |
Protein Variants
EC Number |
Protein Variants |
Comment |
Organism |
---|
1.5.1.3 |
additional information |
incorporation of an alkyne-bearing non-natural amino acid, p-ethynylphenylalanine, into murine dihydrofolate reductase (mDHFR) at Val43, in high cell density cultivation of Escherichia coli, and performance of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation with fluorescent azide dyes to evaluate enzyme compatibility with various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19fold based on the same amount of non-natural amino acid. Enzyme incubation under optimized reaction condition does not lead to any activity loss but allows a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer is efficiently conjugated to enzyme mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. Method evaluation, overview |
Mus musculus |
Inhibitors
EC Number |
Inhibitors |
Comment |
Organism |
Structure |
---|
1.5.1.3 |
DMSO |
irreversibly impairs the enzymatic activity at 30% v/v |
Mus musculus |
|
Natural Substrates/ Products (Substrates)
EC Number |
Natural Substrates |
Organism |
Comment (Nat. Sub.) |
Natural Products |
Comment (Nat. Pro.) |
Rev. |
Reac. |
---|
1.5.1.3 |
7,8-dihydrofolate + NADPH + H+ |
Mus musculus |
- |
5,6,7,8-tetrahydrofolate + NADP+ |
- |
r |
|
Organic Solvent Stability
EC Number |
Organic Solvent |
Comment |
Organism |
---|
1.5.1.3 |
DMSO |
irreversibly impairs the enzymatic activity at 30% v/v |
Mus musculus |
Organism
EC Number |
Organism |
UniProt |
Comment |
Textmining |
---|
1.5.1.3 |
Mus musculus |
P00375 |
- |
- |
Purification (Commentary)
EC Number |
Purification (Comment) |
Organism |
---|
1.5.1.3 |
recombinant His-tagged wild-type mDHFR and modified mDHFR-43pEthF enzymes from Escherichia coli strain XL1-Blue by nickel affinity chromatography |
Mus musculus |
Substrates and Products (Substrate)
EC Number |
Substrates |
Comment Substrates |
Organism |
Products |
Comment (Products) |
Rev. |
Reac. |
---|
1.5.1.3 |
7,8-dihydrofolate + NADPH + H+ |
- |
Mus musculus |
5,6,7,8-tetrahydrofolate + NADP+ |
- |
r |
|
Synonyms
EC Number |
Synonyms |
Comment |
Organism |
---|
1.5.1.3 |
dihydrofolate reductase |
- |
Mus musculus |
1.5.1.3 |
mDHFR |
- |
Mus musculus |
Temperature Optimum [°C]
EC Number |
Temperature Optimum [°C] |
Temperature Optimum Maximum [°C] |
Comment |
Organism |
---|
1.5.1.3 |
25 |
- |
assay at |
Mus musculus |
pH Optimum
EC Number |
pH Optimum Minimum |
pH Optimum Maximum |
Comment |
Organism |
---|
1.5.1.3 |
7.5 |
8 |
assay at |
Mus musculus |
Cofactor
EC Number |
Cofactor |
Comment |
Organism |
Structure |
---|
1.5.1.3 |
NADP+ |
- |
Mus musculus |
|
1.5.1.3 |
NADPH |
- |
Mus musculus |
|