Cloned (Comment) | Organism |
---|---|
expression vectors encoding the full-length zApe and zApe (33-310) proteins in pET15b are constructed using standard subcloning techniques and verified by DNA sequencing. The zApe vectors are transformed into Rosetta (DE3) Escherichia coli, cultures are grown in LB media | Danio rerio |
The wild-type and mutant hApe1 proteins are expressed as GST-fusions in Escherichia coli | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
crystals of zApe (74 mg/ml in 10 mM HEPES, pH 7.5 buffer) are grown by hanging drop vapor diffusion at 20°C using 200 mM ammonium acetate, 100 mM Bis-Tris pH 5.5, 7% PEG 3350, 2% glycerol and 0.8 mM lead acetate as the precipitating solution. The structure is solved at a resolution of 49.15-2.3 A | Danio rerio |
mutant C65A hApe1 protein is diluted to 10 mg/ml in 10 mM HEPES pH 7.5 and then crystallized by hanging drop vapor diffusion using 10 mM MES pH 6.0, 7.5 mM Sm(OAc)3, 4% dioxane, 10-20% PEG 8000 as the precipitating solution at 20°C. The structure is solved at a resolution of 33.0-1.9 A | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
C138A | stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active | Homo sapiens |
C208A | stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active | Homo sapiens |
C296A | stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active | Homo sapiens |
C310A | stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active | Homo sapiens |
C65A | site-directed mutagenesis of a pET28A vector encoding human Ape1 (40-318) is performed, mutant is redox inactive | Homo sapiens |
C93A | stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active | Homo sapiens |
C99A | stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active | Homo sapiens |
T58C | Mutant enzyme is redox active. Site-directed mutagenesis is performed on the full-length zebrafish Ape pET15b vector using Stratagene Quikchange kit and verified by DNA sequencing. The T58C zApe vector is then transformed into Rosetta Escherichia coli (DE3). Stable ovarian cancer Skov-3X cells with the NFkappaB-Luc gene are cotransfected with plasmid pcDNA-mutant and a Renilla luciferase control reporter vector pRL-CMV using lipofectamine. | Danio rerio |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
DNA | Homo sapiens | - |
fragments of DNA | - |
? | |
DNA | Danio rerio | - |
fragments of DNA | - |
? | |
additional information | Homo sapiens | enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others | ? | - |
? | |
additional information | Danio rerio | enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Danio rerio | - |
zebrafish | - |
Homo sapiens | - |
- |
- |
Purification (Comment) | Organism |
---|---|
First step is a Ni-NTA affinity chromatography. Then the Ni-NTA fractions containing zApe are combined and loaded on a pre-equilibrated MonoQ column after dilution to a final NaCl concentration of 50 mM. The protein is eluted using a linear NaCl gradient, and fractions containing the zApe are combined, digested with thrombin, and then subjected again to a final MonoQ column. The purified proteins are concentrated to 50-75 mg/ml. The mutant T58C is purified by using an Q-sepharose column as ion exchanger. | Danio rerio |
Purification is done using the standard glutathione affinity purification protocol. The C65A mutant is purified as follows: cell suspension is centrifuged, supernatant is loaded on a Ni-NTA column and the protein is eluted with a linear imidazole gradient. Fractions containing C65A hApe1 are further purified on an S-Sepharose column with a linear NaCl gradient. | Homo sapiens |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
Cell-based analysis of redox activity for hApe1 Cys-mutants using an in vitro EMSA redox assay in which reduction of the trancription factor AP-1 by hApe1 results in a shifted band. Only C65A hApe1 is found to be redox inactive. | Homo sapiens |
additional information | - |
T58C zApe has near wild-type human Ape1 redox activity in the transactivation assay | Danio rerio |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
DNA | - |
Homo sapiens | fragments of DNA | - |
? | |
DNA | - |
Danio rerio | fragments of DNA | - |
? | |
additional information | enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others | Homo sapiens | ? | - |
? | |
additional information | enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others | Danio rerio | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | C65A hApe1 in solution, determined by size exclusion chromatography | Homo sapiens |
monomer | zApe in solution, determined by gel filtration chromatography | Danio rerio |
Synonyms | Comment | Organism |
---|---|---|
Ape | - |
Homo sapiens |
Ape | - |
Danio rerio |
apurinic/apyrimidinic endonuclease | - |
Homo sapiens |
apurinic/apyrimidinic endonuclease | - |
Danio rerio |
hAPE | - |
Homo sapiens |
hAPE1 | - |
Homo sapiens |
zApe | - |
Danio rerio |