Table 1 Detailed description of cloning, expression and purification of nine cis- and trans-editing domains from P. falciparum.
Enzyme | PlasmoDB ID | Protein length(aa) | Cloning sites | Cloning vector(EMBL) | Expression strain | Induction | Buffer used | Resin used | Purification method |
|---|---|---|---|---|---|---|---|---|---|
Pf-Ed-IRS1 | PF13_0179 | 388–577 | 5′NcoI-3′KpnI | pETM-41 | B834(DE3) | Culture was induced with IPTG (0.5 mM at OD of 0.6) and growth was continued for O/N at 18°C | Lysis buffer-20 mM Tris (7.4), 200 mM Nacl, 10% glycerol, 10 mM βME Elution buffer-10 mM Maltose in lysis buffer | Amylose resin | Superdex S-200 gel filtration chromatography (Amersham Biosciences). |
Pf-Ed-IRS2 | PFL1210w | 551–800 | 5′Ncol-3′Kpnl | pETM-41 | B834(DE3) | Culture was induced with IPTG (0.5 mM at OD of 0.6) and growth was continued for O/N at 18°C | Lysis buffer-20 mM Tris (7.4), 200 mM Nacl, 10% glycerol, 10 mM βME Elution buffer-10 mM Maltose in lysis buffer | Amylose resin | Superdex S-200 gel filtration chromatography (Amersham Biosciences). |
Pf-Ed-LRS | PFF1095w | 367–694 | 5′Ncol-3′KpnI | pETM-41 | B834(DE3) | Culture was induced with IPTG (0.5 mM at OD of 0.6) and growth was continued for O/N at 18°C | Lysis buffer-20 mM Iris (7.4), 200 mM Nacl, 10% glycerol, 10 mM βME Elution buffer-10 mM Maltose in lysis buffer | Amylose resin | Superdex S-200 gel filtration chromatography (Amersham Biosciences). |
Pf-Ed-VRS | PF14_0589 | 254–407 | 5′NcoI-3′KpnI | pETM-11 | BL21-DE3 | Culture was induced with IPTG (0.5 mM at OD of 0.6) and growth was continued for 4 hours at 18°C | Lysis buffer-20 mM Tris (8.0), 500 mM Nacl, 10% glycerol, 10 mM βME Elution buffer-20 mM Immidizaole in lysis buffer | Ni-NTA beads (Qiagen) | Superdex S-75 gel filtration chromatography (Amersham Biosciences). |
Pf-Ed-ARS | PF13_0354 | 998–1234 | 5′NcoI-3′KpnI | pETM-11 | BL21pLysS | Culture was induced with IPTG (1 mM at OD of 0.6–0.8) and growth was continued for O/N at 18°C | Lysis buffer-20 mM Tris (8.0), 500 mM Nacl, 10% glycerol, 10 mM β ME Elution buffer-20 mM Immidizaole in lysis buffer | Ni-NTA beads (Qiagen) | N/A |
PF-Ed-FRS | PF11_0051 | 1–623 | 5′NcoI-3′KpnI | pETM-11 | BL21-DE3 | Culture was induced with (0.5 mM IPTG at O.D. of 0.8) and growth was continued for O/N at 18°C | Lysis buffer-20 mM Tris, 300 mM NaCl, 20 mM Imidaole (pH 8), Elution buffer-increasing concentration of imidazole (upto 500 mM) | Ni-NTA beads (Qiagen) | N/A |
Pf-Ed-TRS | PF11_0270 | 352–528 | 5′NcoI-3′KpnI | pETM-11 | BL21pLysS | Culture was induced with IPTG (1 mM at OD of 0.6–0.8) and growth was continued for O/N | Lysis buffer-20 mM Tris (8.0), 500 mM Nacl, 10% glycerol, 10 mM βME Elution buffer 20 mM Immidizaole | Ni-NTA bea ds (Qiagen) | N/A |
Pf-Ed-PRS | PFL0670c | 1–746 | 5′NcoI-3′KpnI | pETM-30 | B834(DE3) | Culture was induced with IPTG (0.5 mM at OD of 0.6) and growth was continued for O/N at 18°C | Lysis Buffer-20 mM Tris, 300 mM NaCl, Imidazole, 20 mM Imidazole (pH 8). Elution buffer-increasing concentration of imidazole (upto 500 mM) | Ni-NTA beads (Qiagen) | Superdex S-200 gel filtration chromatography (Amersham Biosciences). |
Pf-DTD | PF11_0095 | 1–164 | 5′NcoI-3′KpnI | pET-28a | B834(DE3) | Culture was induced with IPTG (0.5 mM at OD of 0.6) and growth was continued for 5 h at 37°C | Lysis buffer-50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.3 Elution buffer-increasing concentrati on of imidazole (up to 500 mM). | Ni-NTA beads (Qiagen) | Superdex S-75 gel filtration chromatography (Amersham Biosciences). |
