Investigation and development of transient production process for porcine circovirus Type-2 (PCV2) capsid protein in...

Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model "difficult-to-express" virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90β, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other "difficult-to-express" virus capsid proteins in the mammalian cell system.