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The demand for larger quantities of high-purity plasmids continues to increase. Substantial quantities of plasmid DNA are needed to support viral and plasmid-based gene-therapy programmes, including drug delivery, preclinical and clinical studies and production of DNA vaccines. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of plasmid DNA is becoming more widespread. Development of an efficient capture-unit operation for very large plasmid DNA molecules from these large lysate volumes can present a considerable challenge. Use of conventional chromatographic beaded media for plasmid capture is problematic, owing to their restrictive pores, which limits access of the large DNA molecules to only those binding sites on the beads' outer surface. Anion-exchange membranes, on the other hand, have large convective pores that allow the plasmid DNA to readily access all of the membrane's anionic binding sites, even at very high flow rates. The convenience of anion-exchange membranes, configured in ready-to-use capsules, can greatly simplify large-scale plasmid purification strategies. The effectiveness of membrane-based technology for the capture of a pCAT reporter plasmid from large volumes of clarified Escherichia coli lysate was demonstrated. The captured and eluted plasmid pool was found to have been concentrated 10-fold with a reduction in endotoxin of four orders of magnitude.

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A protein methylase III responsible for specifically methylating the cytochrome c in Neurospora crassa was partially characterized by using unmethylated horse heart cytochrome c as a substrate. This enzyme utilizes S-adenosyl-L-methionine as the methyl donor. An analysis of the distribution of [14C]methyl groups in the peptides obtained by chymotrypsin digestion of the enzymically methylated cytochrome c showed that all of the radioactivity could be recovered within a single peak after chromatography. This indicates that the enzyme methylates a specific amino acid sequence within cytochrome c. On hydrolysis of the radioactive chymotryptic peptide, Me-14C-labelled epsilon -N-mono-methyl-lysine, epsilon-N-dimethyl-lysine and epsilon-N-trimethyl-lysine were identified. The enzyme can easily be extracted from the N. crassa mycelial pads and was purified approx. 30-fold.

No abstract