Isolation of plasmid DNA from cell lysates by aqueous two‐phase systems

Abstract

This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.

Extraction and stability of bovine serum albumin (BSA) using cholinium-based Good's buffers ionic liquids

“…In this aspect, aqueous biphasic systems (ABS) are an alternative and scalable technique for the separation and purification of proteins. ABS also allow proteins to maintain their biological activities due to their water-rich environment [5]. In most circumstances, ABS consist of a polymer-rich top phase and a second polymer-or salt-rich bottom phase [5].…”

“…ABS also allow proteins to maintain their biological activities due to their water-rich environment [5]. In most circumstances, ABS consist of a polymer-rich top phase and a second polymer-or salt-rich bottom phase [5]. Amongst the investigated polymers, polyethylene glycol (PEG), polypropylene glycol (PPG) and dextran are frequently used.…”

Scalable Technology to Produce Pharmaceutical Grade Plasmid DNA for Gene Therapy

“…The use of precipitation for the purification of pDNA is reported with the use of natural and chemical precipitants such as calcium chloride salt, after the step of breaking made alkaline to achieve selective precipitation of the cDNA and RNA, obtained as a result, reduction of 50 to 70% of RNA and cDNA, respectively, and the loss of 10% of pDNA. There are also reports of use of other agents which leads to a reduction in volume by concentration and removal of nucleic acids of low molar mass using agents such as isopropanol, polyethylene glycol (PEG) or cetyl trimethylammonium bromide (CTAB) (Lander, 2002;Ribeiro, 2002). Precipitation with ethanol and isopropanol is favourable laboratory scale but not at large scale; it implies a high investment cost.…”

“…The liquid -liquid extraction is another operation that can be used also for the intermediate step of purification using the aqueous two-phase systems (ATPS). So far they have been reported for the purification of pDNA, two-phase systems formed by PEG and salt polymer K2HPO4 (Ribeiro, 2002), where yields are reported 39, 42 and 100% molar mass PEG 300, 600 or 1000 respectively. The two-phase aqueous systems are easily scaled without a noticeable change in the nature and process efficiency, enabling high yields, a continuous process and reducing operational costs in relation to the costs of other conventional operations.…”

Purification of plasmid (pVaxLacZ) by hydrophobic interaction chromatography

“…It is now clear that the demand for large amounts of plasmid DNA will be enormous in view of the potential number of users and the prospect of applying DNA vaccines in veterinary diseases (Little-van den Hurk et al, 2000). Product recovery costs become critical in the overall economics of modern biotechnology processes, and the need to have a process complying with the guidelines issued by regulatory agencies increases the interest in developing methods for the downstream processing of plasmids (Ribeiro et al, 2002). A process for the production of plasmid DNA generally follows the steps of fermentation, primary isolations, and purification .…”