Efficient expression system for human antithrombin III in baby hamster kidney cells

Area of Science:

• Molecular Biology
• Gene Expression
• Mammalian Cell Culture

Background:

• Human antithrombin III (AT III) is a crucial protein for regulating coagulation.
• Efficient expression systems are needed for producing recombinant AT III and its variants.
• The immediate early gene enhancer of human cytomegalovirus (hCMV) is a potent transcriptional control element.

Purpose of the Study:

• To evaluate the hCMV enhancer's efficacy in driving high-level expression of human antithrombin III (AT III).
• To develop a rapid method for establishing stable mammalian cell lines with enhanced recombinant protein production.
• To assess the utility of this system for producing modified AT III molecules.

Main Methods:

• Fusion of the hCMV enhancer to the AT III transcription unit, driven by the Simian virus 40 (SV40) promoter.
• Transient expression assays in baby hamster kidney (BHK) cells to measure AT III synthesis.
• Establishment of stable BHK cell lines using a G418/methotrexate selection protocol.

Main Results:

• The hCMV enhancer increased AT III synthesis by three- to fourfold in transient expression assays.
• Stable BHK cell lines expressing AT III were established within 40-50 days.
• These stable cell lines achieved high expression levels, up to 12 micrograms AT III/10(6) cells/24h.

Conclusions:

• The hCMV enhancer is a powerful tool for significantly enhancing recombinant protein expression in mammalian cells.
• A novel, rapid selection protocol facilitates the generation of high-producing stable cell lines.
• This system offers a versatile platform for the efficient production and screening of recombinant proteins, including modified AT III variants.