Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump.
A parallel chromatographic procedure for the purification of milligram amounts of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quarternary ammonium group with respect to elution and regeneration properties and the 75 cm(2) Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimised. By introducing this step into the protocol, endotoxin levels could be reduced approximately 100-fold to <or=5 EU/mg plasmid. The parallel procedure was set up on a multi-channel peristaltic pump and evaluated with four different vectors (2.7-11.5 kbp). Starting with 5-10 g of E. coli cell paste (wet weight) generally saturated the membrane adsorber, resulting in plasmid DNA yields close to 10 mg.
Syrén
Per-Olof
PO
Global Protein Science and Supply, AstraZeneca R&D Södertälje, SE-15185 Södertälje, Sweden.
Rozkov
Aleksei
A
Schmidt
Stefan R
SR
Strömberg
Patrik
P
D000837:Anion Exchange Resins; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange / Q000295:instrumentation*; D004247:DNA / Q000302:isolation & purification*; D010957:Plasmids
D000837:Anion Exchange Resins; D004247:DNA