Hek293 as a recombinant protein factory: three different approaches for protein production

Abstract

Background Recombinant products have reached the market in diverse areas, including pharmaceutical, veterinary food, pesticides and detergents. Noteworthy, approximately 30 recombinant products in the pharmaceutical sector account for more than 90 per cent of all recombinant product sales. Although microbial systems can be considered an attractive option for expressing certain biopharmaceutical proteins, many biopharmaceutical molecules are too large and complex and need to be expressed in mammalian cells. The most reported cell lines used for industrial processes are CHO, NS0, Sp2/O-Ag14 and HEK293. In particular, the latter has been gaining significant importance in the biopharmaceutical field during the last two decades. While initially employed for adenoviral vector production HEK293 became also one of the preferred cell lines for transient or stable protein expression. This is mainly due to its high transfection efficiency. Meanwhile genetic manipulation also includes new techniques for directing the integration of the foreign gene to highly expressed chromosomal sites e.g. by RMCE. In this work, three strategies for recombinant protein production in HEK293 cells have been compared (Figure 1): (St.1) rAdV production expressing protein of interest, (St.2) stable cell line establishment by random gene transfection or (St.3) site-specific integration using RMCE technology. As protein of interest we used the Cap protein from the capsid of porcine circovirus serotype 2 (CAP-PCV2). This virus is related to post weaning multisystemic wasting syndrome (PMWS), which has major implications for the pig industry worldwide. Materials and methods Strategy 1. CapPCV2 recombinant Adenovirus Viral DNA was isolated from field and the gene of the capsid (CapPCV2) was cloned into Adh5 genome using AdEasyXL kit. Virus and protein production were performed by infecting HEK293-F6 suspension cells [1].