An investigation on E. coli host strain influences and strategies to improve supercoiled plasmid DNA production forgene therapy and vaccination
An investigation on E. coli host strain influences and strategies to improve supercoiled plasmid DNA production forgene therapy and vaccination
复制标题
大肠杆菌宿主菌株影响的研究以及提高基因治疗和疫苗接种超螺旋质粒 DNA 产量的策略
DOI：
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
S. Y. Yau
中科院分区：
文献类型：
--
作者：
S. Y. Yau
The growing demand for quick and effective methods of producing large amounts of plasmid DNA for human therapy and vaccination has increased the practical challenges associated with process optimisation to
improve supercoiled plasmid DNA yields obtained through current
methods. The supercoiled isoform of DNA is the preferred form for use in
gene therapy and vaccination as this isoform is known to produce higher
levels of in vitro and in vivo transgene expression than other forms of
plasmid DNA.
This study was designed to investigate whether different strategies can be
implemented early on in a process to improve supercoiled plasmid DNA
yields obtained upstream, with the view to aid and/or ease further
downstream stages. The main theme investigated is the influence of the
host strain on supercoiled plasmid DNA production. Seventeen strains of
Escherichia coli and three different plasmids were investigated at shake
flask scale, before two strains were selected for scale up to 7L
fermentation scale. The results obtained indicated that the host strain
plasmid combination can heavily influence both the quantity and quality of
plasmid DNA obtained and this behaviour cannot simply be determined by
looking at the host strain genotype. Fermentation runs on the two strains
selected for scale up (BL21 DE3 gWiz and HB101 gWiz) demonstrated
that these two strains scale up very well, maintaining high specific pDNA
yields (1.5mg/L/OD for BL21 DE3 gWiz) and high SC-DNA yields (98% for
HB101 gWiz).
Temperature amplification studies using strains harbouring pUC18 have
shown that although most strain-plasmid combinations yielded more
plasmid at a higher temperature of 40°C, the extent of this increase is
highly influenced by the host strain. Indeed in some cases, such as for the strains ABLE K, W3110, W1485, a higher plasmid yield was obtained at
37°C. However, as the growth rates of these cultures were not measured,
the extent of the accumulation of plasmid DNA due to the effects of the
growth rate and/or temperature during the exponential phase of growth is
unknown at this time. Similarities to what has been reported as
temperature induced runaway plasmid replication have been observed in
this study, although no experiments were conducted to confirm whether
these observations were indeed as result of runaway replication as
defined in the literature.
Potential alternative strategies investigated included implementing
anaerobiosis to test if these conditions can improve supercoiled plasmid
DNA production at fermentation scale, and whether a ‘Quiescent cell
expression system’ (a state where chromosomal replication and
expression is temporarily shut down but residual proteins remain
metabolically active) can be implemented to improve plasmid DNA yields
by redirecting resources away from biomass production. The results
suggest that under the conditions set in this study, these strategies do not
increase plasmid DNA production or the percentage of supercoiled
plasmid obtained.
In conclusion, the results from this investigation have demonstrated that a
highly effective and influential strategy for improving the quality and
quantity of plasmid DNA obtained is the initial choice of the host strainplasmid
combination. Further improvements can then be obtained through
the application of other reported fermentation strategies.
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DOI：
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发表时间：
1999
期刊：
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影响因子：
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作者：
S. Raychaudhuri;Joshua M. Stuart;R. Altman
通讯作者：
S. Raychaudhuri;Joshua M. Stuart;R. Altman
影响因子：
15.9
作者：
Fasbender, A;Lee, JH;Welsh, MJ
通讯作者：
Welsh, MJ
影响因子：
4.1
作者：
Phue, JN;Shiloach, J
通讯作者：
Shiloach, J
DOI：
10.1073/pnas.78.5.2747
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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作者：
STERNGLANZ, R;DINARDO, S;WANG, JC
通讯作者：
WANG, JC