High‐Level Recombinant Protein Production in CHO Cells Using an Adenoviral Vector and the Cumate Gene‐Switch

Abstract

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 degrees 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 degrees 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.

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate‐based assays

“…For many years, there has been an increasing demand in developing efficient production processes for biopharmaceuticals like monoclonal antibodies (Chotteau et al, 2007;Kunert et al, 2000), recombinant proteins (Baker et al, 2001;Gaillet et al, 2007), and viral vaccines (Genzel et al, 2004;Lohr et al, 2009) using animal and human cell lines as cell substrate. However, a major problem that can be seen with almost all the producer cell lines is the highly inefficient use of nutrients.…”

Identification of Human Butyrylcholinesterase Organophosphate-Resistant Variants through a Novel Mammalian Enzyme Functional Screen

“…These features allowed the production of functional infective, yet replication-defective adenoviral vectors. The CHO-cTA-CAR suspension cell line was kindly provided by Dr. Ronald Gilbert (Biotechnology Research Institute, National Research Council, Montreal, Canada) (Gaillet et al, 2007). CHO-CAR suspension cells were cultured in CD-CHO medium (Invitrogen).…”

Why Proteins in Mammalian Cells?

“…It may be necessary to arrange expression of viral receptor protein on the cells to be used for protein production. For example, adenoviruses transduce CHO cells very inefficiently unless a receptor protein is coexpressed in the cells (56). Alternatively, production of the virus may be arranged so that the particles display ligands for receptors on cells they would not ordinarily infect, which is called "pseudotyping."…”