The importance of recombinant protein expression and production for structural and functional protein studies is constantly growing.
Unfortunately, while simple, well-established expression systems (such as E. coli and yeast) exist, a high proportion of proteins relevant for biomedical research cannot be produced in sufficient quality and yields using these systems. Therefore the use of eukaryotic expression systems like insect or mammalian cells is often required. While working with a stable clone is optimal when it comes to large scale production of therapeutic proteins, researchers need higher flexibility and faster methods to produce research-grade (‘tool’) proteins for applications like protein characterisation (eg, crystallisation studies), and functional validation (interaction assays or enzyme assays).
Transient expression enables fast and flexible production of high quality eukaryotic ‘tool’ proteins for academic and drug discovery applications. By using optimized systems, milligram amounts of protein can be produced in just a few days, a significantly faster process than using stable clones or pools. The main barriers to this methodology have been the cost of large scale experiments and the restrictions in applicable cell types and culture characteristics. For instance, typically cell lines in suspension are preferred to adherent clones due to their more convenient handling. For many cell lines in suspension, including HEK293 cells, conventional transfection reagents have very limited efficiency.
Lonza has addressed these issues using its extensive transfection expertise and has developed the XpressNOW Transfection Reagent that allows researchers to produce large amounts of functional protein in the preferred suspension HEK293 cell line with unparalleled cost efficiency.
Materials and methods
Assessment of protein yields
HEK293-F cells (less than passage 30) were routinely passaged every 2-3 days with a seeding density of 3-5 x 105 cells/ml. Prior to transfection, the HEK293 cells were in their logarithmic growth phase at a density of 1 to 1.5 x 106 cells per ml. To ensure maximal transfection efficiency, the cells were in a highly uniform single-cell suspension prior to transfection.
HEK293-F cells were transfected with a vector encoding a 45kD growth factor or a 21kD glycoprotein hormone according to the instructions given in the XpressNOW Manual. Briefly, 12.5 µg DNA was resuspended in PBS to a final volume of 100 µl. In a separate tube, 50 µl of XpressNOW Reagent was combined with 50 µl of PBS.
The DNA was added to the XpressNOW Reagent and mixed gently.
After 5 minutes incubation for complex formation, the mix was added to the cells. The cells were incubated at 37°C (humidified, 5% CO2) for 5 (21kD glycoprotein hormone) or 7 (45kD growth factor) days in a volume of 10 ml on an orbital shaker.
Assessment of cell types
Common HEK293 suspension cell lines (HEK293-F, HEK293 EBNA and HEK293-T) were assessed for transient protein production using either XpressNOW Reagent or another transfection reagent (Reagent F). In each experiment, 1 x 107 cells were prepared as detailed above and transfected with a vector encoding a 45kD growth factor. The transfection procedures were conducted according to the manufacturer’s protocol. The transfected cells were incubated for 7 days in a volume of 10 ml on an orbital shaker in a 37°C incubator with humidified atmosphere of 5% CO2. Protein yields were measured by ELISA.
To assess the reliability of the scale-up procedure, 30, 100 and 300 ml of HEK293 suspension cultures were prepared as detailed above. The cells were subsequently transfected with a vector encoding a 35kD glycoprotein hormone using XpressNOW Reagent. After transfection, cells were incubated on an orbital shaker in a 37°C incubator, with humidified atmosphere of 5% CO2. Protein yields were measured by ELISA at 3 and 5 days incubation.
Following the transfection of HEK293-F, it can be seen that XpressNOW Transfection Reagent facilitates the generation of exceptional amounts of human protein in just a few days. Yields of more than 20 mg/l of the 21kD glycoprotein hormone and the 45kD growth factor were measured by ELISA (Figure 1) after either 5 or 7 days incubation. In other experiments (data not shown), up to 100 mg/l of human cytokine has been produced.
Efficient for a broad variety of suspension HEK293 cells
Previously transfection efficiencies were influenced by the combination of transfection methods with the appropriate cell type. Experiments using XpressNOW Reagent showed good levels of protein yield from different subclones of HEK293 cells. This negates the need for lengthy experiments and changes in preferred clones when using this methodology. The Reagent F tested was obviously optimized for performance with a particular subclone and produces similar amounts of protein to XpressNOW Reagent in this clone, but significantly lower yields were noted in alternative clones.
Reliable scale-up of protein production
The use of transient expression systems has previously been limited by their optimal volume of one litre. The scale-up procedure conducted using XpressNOW Reagent showed that significantly increasing the volume of the culture does not have any detrimental effect on the concentrations of protein produced. XpressNOW Reagent has been optimized to allow reliable scale-up to larger culture volumes and simply requires the transfer of the determined optimal transfection conditions from 25 ml pilot expressions to large scale incubations of 10 L volume or even more.
The new XpressNOW Transfection Reagent provides researchers with an easy, reproducible and affordable method for obtaining high protein yields from transient expression in HEK293 cells. Unlike other methods, it can be scaled up to a 10 L volume and is not limited by a requirement for specific culture media or HEK293 cell type.
By Claire Scholfield, Magda Stosik, Nicole Faust and Volker Vogel, Lonza Cologne AG