Choe W S, Middelberg A P
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, U.K.
Biotechnol Prog. 2001 Nov-Dec;17(6):1107-13. doi: 10.1021/bp010110p.
The direct chemical extraction of recombinant L1 protein (the major capsid protein of human papillomavirus type 16) from the cytoplasm of E. coli HMS174(DE3) has recently been demonstrated at high cell density (to OD(600) = 160) without the use of reducing agent (1). Coextraction of DNA at high concentration prevents direct coupling to postextraction recovery operations including expanded bed adsorption. In this study, spermine is used to selectively precipitate DNA during chemical extraction. Highly efficient and selective DNA precipitation was achieved. An approximate 10-fold increase in the specific spermine concentration (mg of spermine/mg of DNA) was required to precipitate DNA when 8 M urea was added to the extraction buffer. EDTA (3 mM), required for effective chemical extraction, does not significantly inhibit DNA precipitation. Precipitation selectivity was demonstrated in a bovine serum albumin spiking test, with almost complete recovery of the spiked protein. During studies on the direct extraction of L1 protein from cells at OD(600) = 80, high DNA removal efficiency (>85%) and negligible L1 protein coprecipitation were achieved. This selective precipitation technique simply requires the addition of spermine to the chemical extraction buffer and therefore does not increase technique complexity. This modification enhances the method's general applicability and enables direct coupling to downstream recovery units following chemical extraction at high cell and product concentrations.
最近已证明，在不使用还原剂的情况下，可在高细胞密度（OD(600)=160）时从大肠杆菌HMS174(DE3)的细胞质中直接化学提取重组L1蛋白（人乳头瘤病毒16型的主要衣壳蛋白）(1)。高浓度DNA的共提取会妨碍与包括扩张床吸附在内的提取后回收操作直接偶联。在本研究中，精胺用于在化学提取过程中选择性沉淀DNA。实现了高效且选择性的DNA沉淀。当向提取缓冲液中添加8 M尿素时，沉淀DNA所需的特定精胺浓度（精胺毫克数/DNA毫克数）大约增加10倍。有效化学提取所需的EDTA（3 mM）不会显著抑制DNA沉淀。在牛血清白蛋白加标试验中证明了沉淀的选择性，加标的蛋白质几乎完全回收。在对OD(600)=80的细胞直接提取L1蛋白的研究中，实现了高DNA去除效率（>85%）且L1蛋白共沉淀可忽略不计。这种选择性沉淀技术仅需向化学提取缓冲液中添加精胺，因此不会增加技术复杂性。这种改进提高了该方法的普遍适用性，并能够在高细胞和产物浓度下进行化学提取后直接与下游回收单元偶联。
