A simple, robust and highly efficient transient expression system for producing antibodies

详细信息    查看全文

• 作者：Tom Vink ; Maroeska Oudshoorn-Dickmann ; Marcel Roza ; Jelte-Jan Reitsma ; Rob N. de Jong
• 关键词：HEK-293F ; FreeStyle&trade ; 293-F ; HEK-293 ; human embryonic kidney 293 cell line ; CMV ; cytomegalo virus ; SVLT ; the large T antigen of the SV40 virus ; DoE ; design of experiments ; Medium ; FreeStyle&trade ; 293 expression medium ; Optimem ; opti-MEM® ; I reduced-serum medium ; 293Fectin ; 293fectin&trade ; HC ; antibody heavy chain ; LC ; antibody light chain ; p21 ; cyclin-dependent kinase inhibitor 1 ; p27 ; cyclin-dependent kinase inhibitor 1B
• 刊名：Methods
• 出版年：1 January, 2014
• 年：2014
• 卷：65
• 期：1
• 页码：5-10
• 全文大小：491 K

文摘

Transient expression systems in mammalian cells have become the method of choice for producing research quantities of antibodies. Both the speed and yield of the available transient systems and the natural posttranslational modifications favor these systems above expression in lower eukaryotes, prokaryotes or stable cell lines.

We describe an optimized mammalian transient expression system, capable of producing up to 400 mg/L of native secreted antibodies in less than a week. The system is composed of commercially available components and is based on expression in the fast growing suspension cell line, FreeStyle鈩?293-F (HEK-293F).

The method depends on an optimal combination of a gene transfer method, an expression vector and cotransfection with expression enhancing plasmids, encoding the large T antigen of the SV40 virus and the cell cycle inhibitors p21 and p27.

Optimization of all components of the expression system, by experimental design techniques, yielded maximal expression levels (including antibody isotypes IgG1, 2, 3, 4 and Fab fragments of various species).

Expression volumes were scalable from 0.1 ml up to 1.2 L in a simple shaker flask system in animal component free, low protein medium, enabling consistent production of relatively high amounts of a large number of native antibodies.