Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale.

Transient expression of heterologous proteins in mammalian systems is a powerful way to generate protein reagents quickly. However, it has historically suffered from poor yields in comparison to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. Transient methods have been well described for HEK-based systems. In this paper we show the use of a design of experiments (DoE) approach to quickly analyse the effect of a range of different parameters on protein expression from a CHO-based transient system. We show that this system is amenable to a very simple transfection procedure by independent direct addition of DNA and transfection reagent to the culture vessel. In addition we show that expression can be improved by reducing the temperature of the culture conditions post-transfection. The process is demonstrated to be transferrable from 3 ml cultures in deep 24-well plates through cultures in CultiFlask Bioreactors, shake flasks and up to 25 L culture in Wave Bioreactors. Data are shown to illustrate the utility of the system with a number of different classes of protein.

Transient expression, purification and characterisation of human full-length PPARγ2 in HEK293 cells.

Effective anti-diabetic drugs known as thiazolidinediones (e.g. rosiglitazone, pioglitazone) exert their therapeutic effects through their agonistic activity at the peroxisome proliferator-activated receptor gamma (PPARγ). As a multidomain transcription factor, PPARγ forms heterodimers with different retinoid X receptors (RXRs) to modulate target gene expression at the transcriptional level in response to natural or synthetic ligands. Difficulties in producing either of the two major human PPARγ isoforms (PPARγ1 and PPARγ2) as pure full-length proteins in adequate quantity has hindered detailed mechanistic studies of PPARγ and its ancillary protein partners. Here we report an efficient transient expression system to produce recombinant human full-length PPARγ2 protein. The DNA encoding the human full-length PPARγ2 was cloned into a mammalian episomal vector and transiently expressed in human embryonic kidney 293 (HEK293-6E) cells with an expression level of 10mg/L culture. Identity of the purified recombinant PPARγ2 protein was confirmed by mass spectrometry analysis. The purified PPARγ2 protein was active in ligand binding and could be phosphorylated in vitro by Cdk5/p25 at Ser 273. Further studies showed that selected PPARγ modulators inhibited Cdk5-mediated PPARγ2 Ser 273 phosphorylation in vitro. Our results demonstrate the feasibility of producing large quantities of pure and functional human full-length PPARγ2 suitable for drug discovery applications.