提高内毒素去除效果的质粒DNA层析纯化工艺的优化

Enhancement of Removal of Endotoxin by Optimizing Chromatography Procedure for Purification of Plasmid DNA

目的优化质粒DNA分子筛或离子交换层析工艺,提高内毒素去除效果。方法在经精胺沉淀初步纯化的质粒DNA溶液中,加入不同浓度的EDTA(5、10和20mmol/L)或SDS(0.1%、0.2%和0.5%),分别进行Sephacryl S-1000分子筛层析及Source30Q离子交换层析,收集质粒峰,测定内毒素含量。结果用含0.5%SDS的质粒溶液进行分子筛层析,用含10～20mmol/L EDTA或0.1%～0.5%SDS的质粒溶液进行离子交换层析,质粒DNA的内毒素含量均<5EU/mg。结论添加一定浓度的EDTA或SDS可提高质粒DNA分子筛层析或离子交换层析去除内毒素的效果。综合考虑安全性因素,推荐用含10mmol/L EDTA的质粒溶液进行Source30Q离子交换层析。

Objective To optimize the molecular sieve or ion exchange chromatography procedures for purification of plasmid DNA and enhance the removal of endotoxin. Methods After primary purification by spermine precipitation, plasmid DNA was further purified by Source 30Q ion exchange chromatography or Sephacryl S-1000 molecular sieve chromatography in the absence or presence of different concentrations of EDTA （5, 10 and 20 mmol/L） or SDS （0. 1%, 0. 2% and 0. 5%）. The plasmid fractions were collected and tested for residual endotoxin. Results Satisfactory endotoxin removal （residual endotoxin content was less than 5 EU/mg） was achieved by ion exchange chromatography in the presence of 10 - 20 mmol/L EDTA or 0. 1% - 0. 5% SDS, or by molecular sieve chromatography in the presence of 0. 5% SDS. Conclusion The addition of EDTA or SDS at a certain concentration enhanced the removal of endotoxin from plasmid DNA by molecular sieve or ion exchange chromatography. Considering the safety factor, Source 30Q ion exchange chromatography in the presence of 10 mmol/L EDTA was recommended.