McCall Eileen J, Danielsson Anette, Hardern Ian M, Dartsch Christine, Hicks Ryan, Wahlberg Johanna M, Abbott W Mark
Global Protein Science and Supply, Global Sciences and Information, AstraZeneca at Mereside, Alderley Park, Macclesfield, Cheshire, UK.
Protein Expr Purif. 2005 Jul;42(1):29-36. doi: 10.1016/j.pep.2005.03.021. Epub 2005 Apr 11.
Recombinant baculoviruses have proved to be a very useful means to express many proteins over the last 20 years. Since their introduction, there have been a number of significant improvements that have simplified and speeded up the construction of baculoviruses. One of the most commonly used methods relies upon recombination with the baculovirus genome maintained in Escherichia coli. In this paper, we report the conversion of nearly all the steps in this process including the expression testing and purification to a multi-well plate format. This enables a significant increase in the number of constructs that can be processed in a shorter period of time and an order of magnitude increase in the number of expression conditions that can be analysed. A key step in our process is that the transfection is done in suspension rather than adherent cells, which gives a much higher virus titre than in the standard methods.
在过去20年里，重组杆状病毒已被证明是表达多种蛋白质的非常有用的手段。自引入以来，已经有许多重大改进，简化并加速了杆状病毒的构建。最常用的方法之一依赖于与保存在大肠杆菌中的杆状病毒基因组进行重组。在本文中，我们报告了该过程中几乎所有步骤的转化，包括表达测试和纯化，转化为多孔板形式。这使得在更短的时间内能够处理的构建体数量显著增加，并且能够分析的表达条件数量增加了一个数量级。我们方法中的一个关键步骤是转染在悬浮细胞而非贴壁细胞中进行，这比标准方法产生的病毒滴度高得多。
