Transient transfection factors for high-level recombinant protein production in suspension cultured mammalian cells.
The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.
Liu
Chaoting
C
Research and Development, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA. chaoting.liu@invitrogen.com
Dalby
Brian
B
Chen
Weixing
W
Kilzer
Jennifer M
JM
Chiou
Henry C
HC
D000818:Animals; D016466:CHO Cells; D002452:Cell Count; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D004921:Erythropoietin / Q000378:metabolism; D005164:Factor IX / Q000096:biosynthesis; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000096:biosynthesis; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D007668:Kidney / Q000378:metabolism*; D008081:Liposomes / Q000493:pharmacokinetics; D010957:Plasmids / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods*
D016895:Culture Media, Serum-Free; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008081:Liposomes; D011994:Recombinant Proteins; D004921:Erythropoietin; D049452:Green Fluorescent Proteins; D005164:Factor IX