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High‐Level Recombinant Protein Production in CHO Cells Using an Adenoviral Vector and the Cumate Gene‐Switch
Author(s) -
Gaillet Bruno,
Gilbert Rénald,
Amziani Rachid,
Guilbault Claire,
Gadoury Christine,
Caron Antoine W.,
Mullick Alaka,
Garnier Alain,
Massie Bernard
Publication year - 2007
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp060187j
Subject(s) - chinese hamster ovary cell , transduction (biophysics) , internal ribosome entry site , biology , cell culture , microbiology and biotechnology , green fluorescent protein , adenoviridae , adenovirus infection , recombinant dna , protein biosynthesis , gene , virology , messenger rna , translation (biology) , biochemistry , virus , genetics
To facilitate and accelerate the production of eukaryotic proteins with correct post‐translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO‐cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene‐switch transcription system. This cell line is adapted to suspension culture and can grow in serum‐free and protein‐free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO‐cTA‐CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO‐cTA and CHO‐cTA‐CAR using different quantities of AdVs at 37 and 30 °C. Incubation at the latter temperature increased the production of SEAP at least 10‐fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6–7 days post‐infection at 30 °C by transducing CHO‐cTA‐CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large‐scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large‐scale protein production.