Improved methods of cultivation and production of deuteriated proteins from E-coli strains grown on fully deuteriated minimal medium
Improved methods of cultivation and production of deuteriated proteins from E-coli strains grown on fully deuteriated minimal medium
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DOI：
10.1046/j.1365-2672.2003.01866.x
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发表时间：
2003-01-01
影响因子：
4
通讯作者：
Barber, J
中科院分区：
文献类型：
--
作者：
Paliy, O;Bloor, D;Barber, J
目的：目的是开发可靠而经济的细菌生产全氚生物分子的方案。这需要制备耐氚细菌菌株，并了解获得耐氚的生理机制。方法和结果：我们报道了改进的方法，用于在完全氢化的最小培养基上培养大肠杆菌。开发了一种多阶段适应方案；这包括重复培养和选择菌落，并产生高度耐氚的细胞培养。JM109、MRE600和MRE600Rif三株大肠埃希菌均适合在含氢琥珀酸的培养基上生长。这是首次报道了JM109被改编成去氚的最低限度的介质。适应的菌株表现出良好的、稳定的生长速度，并能够用质粒转化。外源蛋白在这些菌株中的表达是可靠的，产量一直很高(100-200mgL(-1))。结论：我们开发了一种新的适应方案，从而获得了三株高度耐氚的大肠杆菌菌株。适应重离子的培养物产生重组化的重组体的高产量。我们认为，大肠埃希氏菌细胞天生就能够在去氢的培养基上生长，但非特异性突变增强了氘的耐受性。因此，平板化和菌落的选择导致了高度耐氚菌株的产生。研究的意义和影响：了解大肠杆菌适应于在氚介质上生长的机制，有助于开发适合高水平生产氚化重组蛋白和其他生物分子的失能耐氚菌株的策略。这对于生物分子的核磁共振和中子散射研究特别重要。
Aims: The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance.Methods and Results: We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium. A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures. Three E. coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium. This is the first report of JM109 being adapted to deuteriated minimal media. The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids. Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l(-1) ). We also show that all E. coli cells are inherently capable of growth on deuteriated media.Conclusions: We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E. coli strains. Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein. We suggest that E. coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance. Thus plating and selection of colonies leads to highly deuterium-tolerant strains.Significance and Impact of Study: An understanding of the mechanism of adaptation of E. coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules. This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.