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Biotechnol Lett (2012) 34:20172022 DOI 10.1007/s10529-012-0994-4
ORIGINAL RESEARCH PAPER
Plasmid size can affect the ability of Escherichia coli to produce high-quality plasmids
Junlin Yang Yong Yang
Received: 31 March 2012 / Accepted: 22 June 2012 / Published online: 11 July 2012 Springer Science+Business Media B.V. 2012
Abstract Large molecular weight plasmids are often used in gene therapy and DNA vaccines. To investigate the effect of plasmid size on the performance of Escherichia coli host strains during plasmid preparation, we employed E. coli JM109 and TOP10 cells to prepare four plasmids ranging from 4.7 to 16.8 kb in size. Each plasmid was extracted from JM109 and TOP10 cells using an alkaline lysis mini-preparation method. However, when commercial kits were used to extract the same plasmids from JM109 cells, the large molecular weight plasmids substantially degraded, compared with their smaller counterparts. No degradation was observed when the four plasmids were extracted from E. coli TOP10 cells using the same commercial kit. We conclude, therefore, that the performance of E. coli in high quality plasmid preparations can be affected by plasmid size.
Keywords endA Host strain Plasmid preparation
Plasmid size
Introduction
Plasmid DNA has been widely used as a non-viral vector to develop safe and effective gene therapies and DNA vaccines for many infectious, acquired, and genetic diseases. Recently, DNA-based gene therapies for the treatment of intermittent claudication and critical limb ischemia have undergone phase III clinical trials (Maulik 2009). Because plasmid DNA can be delivered in the form of a vaccine that elicits immune responses, such vaccines hold promise for the treatment of life threatening viral infections such as AIDS, Ebola, and hemorrhagic fever, and for cancer (Phue et al. 2008; Kutzler and Weiner 2008).
Many large molecular weight plasmids have been designed to accommodate large genes, tissue-specic expression regulatory elements, or multiple immuno-logical protein genes (Levy et al. 1999). To obtain high-quality plasmid DNA, however, has necessitated substantial modications of the E. coli host strains that are routinely used for plasmid propagation. For example, the E. coli genome has been reduced in size to remove mobile elements such as insertion sequences, whilst the endA single gene knockout is thought to prevent plasmid degradation during host cell lysis (Bower and Prather 2009). However, to the best of...