THE PROCESSING OF A PLASMID-BASED GENE FROM ESCHERICHIA-COLI - PRIMARY RECOVERY BY FILTRATION

We describe the primary recovery of plasmid DNA from alkaline lysis mi xtures using a nutsche filter operated under pressure. Six different f ilter cloths constructed of polypropylene, polyester and stainless ste el were tested, with pore sizes ranging from 5-160 mu m. Both pore siz e and the material of the filter membranes employed in filtration expe riments exerted considerable impact on the purity and yield of the pla smid DNA. The greatest degree of solids extrusion, shearing of chromos omal DNA and subsequent contamination of the filtrate was observed wit h the 160 mu m polyester filter. The best compromise was obtained with a 5 mu m polypropylene cloth. For an alkaline lysis mixture containin g 101 g wet weight solids per litre, filtration through this cloth pro ceeded at an average rate of 22.5 cm h(-1). Virtually complete removal of solids (99.4%) and protein (96.8%) was achieved, with a 8.2-fold p urification of plasmid DNA at the expense of a 33% loss in yield. The filtration performance of this membrane was further modified by precoa ting with diatomaceous earths of different permeabilities (0.07-1.2 da rcies). The finest filter aid resulted in very pure plasmid DNA (65%), complete suspended solids removal and < 1% of the original protein re maining in the filtrate. However, the plasmid yield was only 30%, the processing rate was markedly reduced (8.2 cm h(-1)), and some losses o f plasmid DNA, due to adsorption on to the diatomaceous earth, were al so observed (5.7%).