碱性裂解过程中染色体DNA的生成及反胶束萃取去除

Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction.

作者信息

Tschapalda Kirsten, Streitner Nadine, Voss Carsten, Flaschel Erwin

机构信息

Chair of Fermentation Engineering, Faculty of Technology, Bielefeld University, 33501 Bielefeld, Germany.

出版信息

Appl Microbiol Biotechnol. 2009 Aug;84(1):199-204. doi: 10.1007/s00253-009-2088-3. Epub 2009 Jun 27.

DOI:10.1007/s00253-009-2088-3

PMID:19562335

Abstract

The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g(-1) of plasmid DNA with almost complete plasmid recovery.

摘要

通过高度可扩展的纯化技术从药用质粒DNA中分离结构相关杂质是生化工程面临的一项挑战。除了RNA、蛋白质和脂多糖外，还必须去除质粒复制宿主的染色体DNA。在此，我们描述了反胶束萃取在从质粒DNA中分离染色体DNA方面的应用。通过应用不同的碱裂解程序，生成了具有不同量染色体DNA的细菌裂解物。一个反胶束萃取步骤使我们能够将这种杂质的浓度降低到低于质粒DNA所需的50 mg g(-1)水平，同时几乎完全回收质粒。