Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography - A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography. - Bio

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Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography
A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography.


BioPharm International


Production of pIDKE2

E. coli DH10B cells harboring pIDKE2, a plasmid for DNA immunization expressing the first 650 aa of the HCV polyprotein from a genotype 1b-Cuban isolate, were grown over-night, at 37 °C, in 100-mL shake flasks containing 50 μg kanamycin/mL, at 250 rpm.7 The engineered E. coli was cultured using a complex media designed with a 50 g/L of yeast-extract concentration in fed-batch mode to produce pIDKE2 plasmid.

Lysis and Plasmid Recovery

Bacteria cell paste (typically 200 g) was suspended in 2.4 L of 61 mM glucose, 10 mM Tris-HCL, 50 mM EDTA pH 8. Lysis was performed by adding 2.4 L of 0.1 M NaOH and 1% w/v SDS, followed by incubation for 10 min at 4 °C. The lysate was neutralized with 2.4 L of 3 M potassium acetate for 10 min at 4 °C. Precipitated material including cell debris, most chDNA, and proteins were removed by centrifugation at 3,000 g for 10 min. The clarified cell 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 maintaining constant transmembrane pressure (TMP) of 0.8 bar at 4 °C, using Sartocon Slice equipment from Sartorius. The lysate was loaded on Sepharose CL 4B in BPG 113 column to remove RNA using a 20 mM Tris-HCL, 3 mM EDTA, and 1.5 M (NH4)2SO4 pH 7.2 buffer. The pDNA fraction was purified using POROS R1 50 reverse-phase media and the elution was carried out in acetonitrile at 10% v/v. The DNA polishing step was carried out on Sephacryl S-1000 media in formulation buffer: 20 mM NaH2PO4–Na2HPO4; 1 mM EDTA; and 30 mM NaCl pH 6.7 ± 0.2. Fractions containing more than 90% supercoiled plasmids were pooled and concentrated using PEG 8000 and NaCl, following methods published by Lis and Schleif.8

Analytical Methods

Flow-through and eluted fractions were precipitated by 1 volume of 2-propanol for 15 min on ice and centrifuged at 14,000 x g for 10 min at 4 °C. The pellet was washed with 70% ethanol and centrifuged again at 4,000 x g for 10 min. The pellet was air-dried for 5–10 min and dissolved in 50 μl of 10 mM Tris-HCl pH 8.5. Fractions prepared as previously described were tested for restriction digestion analysis by transformation experiments and analyzed by 0.8% agarose gel electrophoresis. Yield and pDNA purity were determined by measuring absorbance at 260 (A260) and 280 (A280) and expressed as A260/A280 ratio in a low-salt buffer. As a positive control, supercoiled pDNA purified by the Horn method was used.9


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