Effects of growth medium selection on plasmid DNA production and initial processing steps
Effects of growth medium selection on plasmid DNA production and initial processing steps
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DOI：
10.1016/s0168-1656(99)00187-x
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发表时间：
2000-01-21
影响因子：
4.1
通讯作者：
Keshavarz-Moore, E
中科院分区：
文献类型：
--
作者：
O'Kennedy, RD;Baldwin, C;Keshavarz-Moore, E
将含有质粒pSV β的重组大肠杆菌培养物在三种培养基配方中生长，以评估它们对用于基于质粒的基因治疗的超螺旋质粒DNA生产特性的影响。发现含有酪蛋白氨基酸的半限定培养基（SDCAS）比含有大豆氨基酸的类似培养基（SDSOY）或Luria-Bertani培养基（LB）支持更高的细胞密度和更高的质粒稳定性。在LB和SDCAS培养基上生长的细胞之间观察到细胞收获特性、质粒DNA初级回收、质粒DNA产量和质量的差异。在SDCAS培养基上生长的细胞在收获后比在LB培养基中生长的细胞更难重悬，并且对碱裂解不太敏感。SDCAS的质粒DNA含量主要是超螺旋的，并且比LB培养基上生长的细胞的质粒DNA提取物受到染色体DNA的污染更少。假设培养基的不同碳氮比（C：N）可能是改变细胞壁多糖组成的原因，导致细胞收获和裂解特性发生变化。结果表明，改变SDCAS培养基的C：N比在1.21：1和12.08：1之间，细胞壁多糖组成或细胞对化学裂解或物理破碎的敏感性没有改变。SDCAS培养基的C：N比增加10倍，质粒DNA产量增加10倍。(C)2000 Elsevier Science B.V.保留所有权利。
Cultures of recombinant Escherichia coli containing the plasmid pSV beta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium. (C) 2000 Elsevier Science B.V. All rights reserved.