Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type mDHFR and modified mDHFR-43pEthF enzymes in Escherichia coli strain XL1-Blue | Mus musculus |
Protein Variants | Comment | Organism |
---|---|---|
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 | Comment | Organism | Structure |
---|---|---|---|
DMSO | irreversibly impairs the enzymatic activity at 30% v/v | Mus musculus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
7,8-dihydrofolate + NADPH + H+ | Mus musculus | - |
5,6,7,8-tetrahydrofolate + NADP+ | - |
r |
Organic Solvent | Comment | Organism |
---|---|---|
DMSO | irreversibly impairs the enzymatic activity at 30% v/v | Mus musculus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mus musculus | P00375 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type mDHFR and modified mDHFR-43pEthF enzymes from Escherichia coli strain XL1-Blue by nickel affinity chromatography | Mus musculus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
7,8-dihydrofolate + NADPH + H+ | - |
Mus musculus | 5,6,7,8-tetrahydrofolate + NADP+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
dihydrofolate reductase | - |
Mus musculus |
mDHFR | - |
Mus musculus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Mus musculus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | 8 | assay at | Mus musculus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Mus musculus | |
NADPH | - |
Mus musculus |