Plasmid Membrane Chromatography
Qiagen column:
Qiagen resins allow a tight binding of
pDNA molecules. Thus, while at lower ionic strength (<1.0 M
NaCl) the proteins and RNA are washed away, pDNA elutes
only at high salt conditions (1.6 M NaCl, pH 7.0)
The bacterial cell paste (typically 200 g) was re-suspended in 2.4 L of a buffer containing 61 mM glucose, 10 mM Tris–HCL, and 50 mM EDTA, at pH 8. Bacteria were lysed using a previously described procedure.4 The clarified lysate was concentrated five times on a tangential flow filtration (TFF) system (nominal molecular weight cutoff 100,000 kDa, 0.1 m2 ). All experiments were conducted keeping a constant transmembrane pressure (TMP) of 0.8 bar at 4 °C, using Sartocon Slice equipment (Sartorius-Stedim Biotech). To remove RNA, the concentrated clarified lysate was loaded on Sepharose CL 4B in a BPG 113 column equilibrated with 20 mM Tris–HCL, 3 mM EDTA, and 1.5 M (NH4)2 SO4, at pH 7.2.
Plasmid capture and elution using the Sartobind membrane was carried out after buffer exchange on a G25 matrix using 25 mM Tris, 10 mM EDTA, and 0.5 M NaCl, at pH 8 and a rate of 50 mL/min, to reach a conductivity of 45 ms/cm. The pDNA fraction was pumped at 150 mL/min to the membrane holder 91-D-01K-15-03. After loading the feeding solution, the membrane was rinsed with a pH 8 buffer containing 25 mM Tris, 10 mM EDTA, and 0.7 M NaCl. Bound plasmid DNA was eluted with a buffer solution containing 25 mM Tris, 10 mM EDTA, and 1.2 M NaCl at pH 8. Finally, the membrane was regenerated using 0.5 M NaOH. The pDNA polishing was achieved on Sephacryl S-1000 media on a formulation buffer of 20 mM NaH2PO4–Na2HPO4, 1 mM EDTA, and 30 mM NaCl at pH 6.7, using a 50X K column (Amersham BioSciences) at 4.8 mL/min. Fractions containing more than 89% supercoiled pDNA were processed by TFF until reaching a concentration of 2 mg/mL as determined by the absorbance at 260, using a VivaFlow 200 system (nominal molecular weight cutoff 100,000 kDa, 200 cm2 ). The final pDNA was filtered (0.22 μm) before further analyses were performed.