Cloned (Comment) | Organism |
---|---|
vector pET30-EK/LIC, expression in Escherichia coli | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
additional information | chimeras of CCA-adding enzyme and PAP were constructed | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00039 | - |
oligo(A)12 | incubation 30 min at 30 °C | Escherichia coli | |
0.13 | - |
ATP | incubation 30 min at 30 °C | Escherichia coli | |
0.27 | - |
CTP | incubation 30 min at 30 °C | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
The wild-type poly(A) polymerase, as well as deletion variants are expressed in Escherichia coli BL21(DE3) (Novagen). Freshly transformed cells are grown at 28-37°C in 700 ml LB medium containing 0.030 mg/ml kanamycin and 0.033 mg/ml chloramphenicol. At mid-log phase (A600 = 0.6), expression is induced by the addition of IPTG to a final concentration of 0.200 mM. After 3-5 hr of incubation at 28-37°C, cells are harvested by centrifugation and lysed by lysozyme treatment and sonication in ice-cold buffer I (20 mM Tris/HCl pH 7.6, 0.5 M NaCl, 5 mM imidazole and 0.75 mg/ml lysozyme). After centrifugation for 30 min at 25000 xg and 4°C, the proteins in the supernatant are purified by FPLC on a 5 ml HiTrap Chelating Sepharose column (Amersham Biosciences) and eluted with 500 mM imidazole. Fractions containing the enzymes as determined by SDS-PAGE are pooled, dialyzed against buffer II (20 mM Tris/HCl pH 7.6, 0.5 M NaCl, 5 mM MgCl2 and 10% glycerol). All proteins are stored in the presence of 40% (v/v) glycerol at -20°C. | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + oligo(A)12 | - |
Escherichia coli | diphosphate + oligo(A)13 | - |
? | |
ATP + RNAn | - |
Escherichia coli | diphosphate + RNAn+1 | - |
? | |
CTP + RNAn | - |
Escherichia coli | diphosphate + RNAn+1 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PAP | - |
Escherichia coli |
poly(A) polymerase | - |
Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.43 | - |
CTP | incubation 30 min at 30 °C | Escherichia coli |